Methods, compositions and systems for microfluidic assays

ABSTRACT

Provided herein, among other aspects, are methods and apparatuses for analyzing particles in a sample. In some aspects, the particles can be analytes, cells, nucleic acids, or proteins and contacted with a tag, partitioned into aliquots, detected by a ranking device, and isolated. The methods and apparatuses provided herein may include a microfluidic chip. In some aspects, the methods and apparatuses may be used to quantify rare in a sample, such as cancer cells and other rare cells for disease diagnosis, prognosis, or treatment.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.14/903,012, filed Jan. 5, 2016, which is a National Stage Entry ofInternational Application No. PCT/US2014/045094, filed Jul. 1, 2014,which claims the benefit of U.S. Provisional Application Nos.61/843,252, filed Jul. 5, 2013 and 61/894,788, filed Oct. 23, 2013,which applications are incorporated herein by reference in theirentirety for all purposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under CA147831 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND

Circulating tumor cells (CTCs) are shed into the bloodstream from theprimary tumor and are an important aspect of cancer metastasis. CTCshave been detected in many different types of cancer, such as breast,lung, prostate and pancreatic cancers. The number of CTCs directlycorrelates with the clinical outcome in metastatic patients, providingvaluable prognostic information that can be helpful to manage clinicalcare.

SUMMARY

Described herein are methods, apparatuses, systems and devices forisolating and analyzing particles and performing assays.

In various aspects, methods are provided for identifying a plurality ofmarkers present on an analyte within a fluid, wherein the methodcomprises: (a) detecting a signal from a first tag using a source ofradiation, wherein the first tag is attached to a first structure thatbinds to a first marker on the analyte; (b) partitioning the analytebased on the presence of the first tag; (c) reducing the intensity ofthe signal of the first tag; (d) contacting the analyte with a secondstructure that binds to a second marker, wherein the second structure isattached to a second tag; and (e) detecting the second tag.

In various aspects, methods are provided for detecting a plurality ofmarkers present on an analyte, the method comprising: contacting theanalyte with a first tag, wherein the analyte comprises a first markerand the first tag has an affinity for the first marker; detecting afirst signal emitted by the first tag, wherein the presence of the firstsignal indicates the presence of the first marker; partitioning a fluidcomprising the analyte based on the presence of the first signal;reducing the intensity of the first signal; contacting the analyte witha second tag, wherein the analyte comprises a second marker and thesecond tag has an affinity for the second marker; and detecting a secondsignal emitted by the second tag, wherein the presence of the secondsignal indicates the presence of the second marker.

In various aspects, methods are provided for isolating cells from asample comprising a first cell type and a second cell type, the methodscomprising: (a) introducing the sample into a microfluidic chip via aset of tubing wherein the microfluidic chip comprises (i) at least onechannel fluidly connected to the set of tubing; (ii) a detectorconfigured to detect signals of cells within the at least one channel;and (iii) at least one chamber fluidly connected to the at least onechannel; (b) flowing a portion of the sample past the detector; (c)using the detector to detect the presence or absence of the first celltype within the portion of the sample; (d) if the first cell type isdetected within the portion of the sample, directing an aliquot of thesample into the chamber, wherein the aliquot comprises the first celltype; and (e) repeating steps (b), (c), and (d), thereby isolatingmultiple aliquots in the chamber such that the chamber comprises greaterthan 80% of a total number of first cell types within the sample andless than 5% of a total number of second cell types within the sample.

In various aspects, methods are provided for isolating cells from asample, the methods comprising: (a) introducing the sample into amicrofluidic chip, wherein the sample comprises a first cell type and asecond cell type, and wherein the microfluidic chip comprises: achannel; a detector configured to detect a signal emitted within thechannel; a chamber in fluidic communication with the channel; (b)flowing a portion of the sample through the channel, wherein the portioncomprises a plurality of the first cell type, a plurality of the secondcell type, or a combination thereof; (c) detecting the presence orabsence of the first cell type within the portion using the detector;(d) directing the portion into the chamber if the first cell type ispresent within the portion; and (e) repeating (b), (c), and (d) asufficient number of times such that the chamber comprises more than 80%of the total number of the first cell type present within the sample andless than 5% of the total number of the second cell type present withinthe sample.

In various aspects, apparatuses are provided for partitioning cellsexpressing a specific biomarker profile from a sample derived from afluid, wherein: the apparatuses comprise a set of tubing connected to amicrofluidic chip that has at least one channel and a chamber; and theapparatuses are capable of isolating the cells in the chamber, wherein,after isolation, the chamber comprises greater than 80% of the totalpopulation of cells in the sample expressing the specific biomarkerprofile and wherein, after isolation, the chamber comprises less than 5%of the total population of cells in the sample expressing a differentbiomarker profile.

In various aspects, methods are provided for identifying a plurality ofmarkers present on an analyte, wherein the methods comprise: (a)partitioning a plurality of analytes by flowing the analytes over asubstrate comprising a plurality of micro-cavities or micro-patches,wherein the majority of micro-cavities or micro-patches are capable ofcontaining not more than one analyte and wherein the micro-cavities ormicro-patches are located in a microfluidic device; (b) in themicro-cavities or micro-patches, contacting each analyte with a firststructure that is capable of binding to a first marker, wherein thefirst structure is connected to a first tag; (c) detecting a signal fromthe first tag; (d) reducing the level of the signal of the first tag;(e) contacting the analyte with a second structure that binds to asecond marker, wherein the second structure is connected to a secondtag; and (f) detecting the second tag.

In various aspects, methods are provided for detecting a plurality ofmarkers present on an analyte, the methods comprising: isolating ananalyte in a micro-cavity or in a micro-patch by flowing a fluid over asubstrate comprising the micro-cavity or micro-patch, wherein the fluidcomprises the analyte; contacting the analyte with a first tag, whereinthe analyte comprises a first marker, and wherein the first tag has anaffinity for the first marker; detecting a first signal emitted by thefirst tag, wherein the presence of the first signal indicates thepresence of the first marker; reducing the intensity of the firstsignal; contacting the analyte with a second tag, wherein the analytecomprises a second marker, and wherein the second tag has an affinityfor the second marker; and detecting a second signal emitted by thesecond tag, wherein the presence of the second signal indicates thepresence of the second marker.

In various aspects, systems are provided for detecting a particle in afluid sample, the systems comprising: a microfluidic chip comprising aninput channel, a first output channel, a second output channel, and adirectional flow channel; a valve, wherein the valve is separable fromthe microfluidic chip, and wherein: the valve regulates the flow of afirst fluid in the directional flow channel; and the flow of the firstfluid in the directional flow channel directs the flow of a second fluidfrom the input channel to the first output channel, the second outputchannel, or a combination thereof; a detector configured to detect asignal emitted from a portion of the second fluid in the input channel;and a processor configured to: assign a value to the portion based onthe signal; and operate the valve. In some aspects, the valve is anelectro-actuated valve.

In various aspects, systems are provided for detecting a particle in afluid sample, the system comprising: (a) a microfluidic chip comprisingat least one sample input channel, at least one directional flowchannel, and at least two output channels, wherein the at least onedirectional flow channel intersects the sample input channel; (b) anelectro-actuated valve that is located on a device that is not part ofthe microfluidic chip, wherein the electro-actuated valve controls theflow of a fluid by controlling an input channel that intersects at leastone directional flow channel or at least one of the at least two outputchannels; (c) at least one detector capable of detecting one or moreanalytes in an aliquot of the fluid sample; and (d) a processor capableof assigning a value to the aliquot based on the presence, absence,identity, composition, or quantity of analytes in the aliquot, whereinthe processor is in communication with the detector and theelectro-actuated valve.

In various aspects, methods are provided for isolating an aliquot of afluid sample within a microfluidic chip, wherein the aliquot comprises arare particle, the methods comprising the steps of: (a) detecting thepresence or absence of the rare particle in the aliquot; (b) assigning avalue to the aliquot based on the presence or absence of the rareparticle; and (c) directing the flow of the aliquot based on theassigned value by opening an electro-actuated valve, wherein theelectro-actuated valve is located on a device that is external to themicrofluidic chip. In some aspects, the microfluidic chip comprises asample input channel, at least two output channels, and at least onedirectional flow channel, and wherein the electro-actuated valvecontrols the flow of fluid within the directional flow channel.

In various aspects, devices are provided for detecting a rare particlein a fluid sample, the devices comprising: an input channel; a firstoutput channel; a second output channel; a detector configured to detectthe presence or absence of a particle in a portion of the fluid sample;a mechanism for directing the flow of the portion from the input channelto the first output channel, the second output channel, or a combinationthereof based on the presence or absence of the particle; and a filterin fluidic communication with the first output channel.

In various aspects, devices are provided for detecting a rare particlein a fluid sample, the devices comprising: (a) at least one sample inputchannel; (b) at least two output channels, wherein at least one of thetwo output channels is in fluidic communication with an array ofapertures; (c) at least one detector capable of detecting one or morerare particles in an aliquot of the fluid sample; and (d) a mechanismfor sorting the one or more rare particles by directing the flow ofaliquots containing the one or more rare particles through a firstoutput channel.

In various aspects, integrated systems are provided for performing anassay, the systems comprising: a fluid sample comprising a particle,wherein the particle comprises a marker; an input channel; a firstoutput channel; a second output channel; a first detector configured todetect the presence or absence of the particle in a portion of the fluidsample, the portion disposed within the input channel; a mechanism fordirecting the flow of the portion from the input channel to the firstoutput channel based on the presence or absence of the particle; amicro-cavity in fluidic communication with the first output channel andconfigured to trap the particle; and a second detector configured todetect the presence or absence of the marker in the micro-cavity.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings of which:

FIG. 1 is an overview of immunostaining and photobleaching using afluidic microfluidic chip according to an aspect of the presentdisclosure.

FIG. 2 is an overview of immunostaining and photobleaching using theeDAR apparatus according to an aspect of the present disclosure.

FIG. 3 is an overview of immunostaining and photobleaching using asingle-analyte array according to an aspect of the present disclosure.

FIG. 4 is an overview of eDAR according to an aspect of the presentdisclosure.

FIG. 5 illustrates an example of the process flow for microfabricationof the eDAR microfluidic chip according to an aspect of the presentdisclosure.

FIG. 6 illustrates eight exemplary hydrodynamic sorting schemes.

FIG. 7 shows the microfluidic chip and hydrodynamic switching scheme ofensemble eDAR according to an aspect of the present disclosure.

FIG. 8 depicts an example of the switching time for the current fluidicscheme recorded by high speed camera according to an aspect of thepresent disclosure.

FIG. 9 shows the characterization and analytical performances of eDARaccording to an aspect of the present disclosure.

FIG. 10 shows the microslits and multicolor fluorescence imaging ofcaptured CTCs according to an aspect of the present disclosure.

FIG. 11 depicts the general structure of the “dual-capture” eDARaccording to an aspect of the present disclosure.

FIG. 12 shows bright field images of the three status of the blood flow.

FIG. 13 provides a general scheme and procedure of the sequentialimmunostaining and photobleaching tests according to an aspect of thepresent disclosure.

FIG. 14 shows sequential immunostaining and photobleaching results forsix cancer cells trapped on an eDAR microfluidic chip according to anaspect of the present disclosure.

FIG. 15 depicts an example of photobleaching curves for the MCF-7 cellslabeled with anti-EpCAM-PE that are exposed to different powers of thelight source according to an aspect of the present disclosure.

FIG. 16 shows fluorescence images of the four cancer cells captured oneDAR with Her2⁺/MUC1⁻ character according to an aspect of the presentdisclosure.

FIG. 17 is a diagram of Single-analyte array according to an aspect ofthe present disclosure.

FIG. 18 is a schematic of a device with trapping densities anddimensions according to an aspect of the present disclosure.

FIG. 19 depicts the parallel flow resistance trap according to an aspectof the present disclosure.

FIG. 20 is a schematic of the procedure used to build some of thedevices described in the present disclosure.

FIG. 21 is an example of a microfabrication method which can be used toproduce the parallel flow resistance trap according to an aspect of thepresent disclosure.

FIG. 22 depicts the steps by which the serial-flow resistance trap andparallel flow resistance trap can collect, discretize, and read outbiologically derived samples according to an aspect of the presentdisclosure.

FIG. 23 shows a detection and read-out scheme for arrays of micro-wellsand side chambers based on brightfield and fluorescence microscopyaccording to an aspect of the present disclosure.

FIG. 24 illustrates the sequence for trapping an array of biologicalparticle/cell for analysis and release according to an aspect of thepresent disclosure.

FIG. 25 shows the distribution of 15 control samples and 10 pancreaticcancer samples that can be analyzed by the method reported hereinaccording to an aspect of the present disclosure.

FIG. 26 shows a CTC cluster with low epithelial cell-adhesion marker(EpCAM) expression from a pancreatic cancer sample according to anaspect of the present disclosure.

DETAILED DESCRIPTION OF THE DISCLOSURE

This disclosure provides methods, systems and devices for detecting,separating and analyzing particles (e.g., cells) in a fluid sample(e.g., blood), often with the use of a microfluidic apparatus. Theparticles can be rare particles (e.g., rare cells). Many of themicrofluidic apparatuses provided herein can be used to perform EnsembleDecision Aliquot Ranking (eDAR) on a fluid sample, which permitsanalysis of the fluid sample after the fluid sample is divided intoaliquots.

In some aspects, an eDAR apparatus can be used to (i) detect thepresence or absence of a rare particle (e.g., rare cell) in an aliquotof the fluid sample, (ii) rank the aliquot according to the presence orabsence of a rare particle, for example by assigning a term such asnon-zero or null to the aliquot; and (iii) direct the flow or collectionof the aliquot based on the assigned ranking using a scheme such as ahydrodynamic switching scheme. The aliquots can be a portion of thetotal volume of a fluid sample to be analyzed. In some aspects, analiquot occupies a three-dimensional space and the particles within thealiquot can be distributed randomly.

In some aspects, this disclosure provides apparatuses and methods forperforming eDAR on a fluid sample with a very high efficiency,sensitivity and/or recovery rate. For example, the eDAR apparatusprovided herein can recover greater than 95% of a particular rare celltype from a fluid sample (e.g., blood, whole blood, urine, etc.). TheeDAR apparatus can function with a less than 10%, less than 5%, or even0% false positive rate. The false positive rate can be over a number ofsamples, such as greater than 10 samples, or greater than 15 samples.The speed of the eDAR apparatus can also be very fast. For example, anentire sample can be processed in less than 25 minutes, less than 20minutes, etc.

Often, the eDAR apparatus provided herein comprises a microfluidic chipcontaining (a) channels; (b) chambers; (c) filters; (d) detectors,and/or (e) valves. The microfluidic chip can be part of a larger systemthat also includes (a) vessels for holding buffers; (b) off-chip valves(e.g., solenoid valves); (c) light sources and detectors; (d) tubing;(e) sample ports; (f) digital processors and/or other features. Thefilters and microslits or micro-apertures (e.g., array of microslits)can be used to further purify a sample. Herein, the terms “microslit”and “micro aperture” are used interchangeably, and microslits or microapertures can also include an array of posts where the inter-postspacing is used for carrying out filtration. The inter-post spacing canbe uniform or variable. In some aspects, a sample is introduced to aneDAR apparatus, which then performs active sorting of the cells by ahydrodynamic switching scheme. The hydrodynamic switching scheme caninclude channels with a particular geometry, as described furtherherein.

In some aspects, this disclosure provides methods and apparatuses forcapturing more than one type of analyte (e.g., rare cell), or more thanone subpopulation of analytes (e.g., rare cells) in a population ofanalytes (e.g., rare cells) within a sample. A sample can be labeledwith a plurality of detection reagents so that a plurality of analytes(e.g., rare cells) are detected. For example, the sample can be a mixedsample and contain a mixed population of rare cells. The mixedpopulation of rare cells can comprise an epithelial cell and amesenchymal cell, amongst other cell types. Within the plurality ofdetection reagents, one detection reagent can bind to an epithelialmarker on the epithelial cell, while a different reagent binds to amesenchymal marker on the mesenchymal cell.

The mixed sample can be introduced into the microfluidic chip apparatusat an input channel. Side channels on the apparatus can be used tocontrol the hydrodynamic switching of the flow of the mixed sample. Theflow of the fluid can be controlled by two solenoids so that theplurality of analytes are separated into two different regions of themicrofluidic chip. In some aspects, the analytes are further purified onthe microfluidic chip, such as by passing the fluid through a filter oran array of microslits. The analytes can be collected at the filter oron the array of microslits.

This disclosure further provides a method for a staining and washingsystem that can be coupled with eDAR, or that can be used with othermicrofluidic devices. The method of the staining and washing system canbe in-line. The in-line staining and washing methods provides for anautomated process of isolating individual analytes (e.g., cells) anddetecting biomarkers. The method can also reduce the amount of detectionreagents (e.g., antibodies) needed to detect different markers on ananalyte. The method can further minimize the dead volume of the system.Additionally, the apparatus can include a mechanism to avoidintroduction of air bubbles into the apparatus.

eDAR can be coupled with downstream methods of characterization andanalysis. The methods can include cellular and molecular analysis ofrare cells. In some aspects, immunostaining can be used to determineexpression of certain biomarkers on rare cells. A semi-automated methodand system for immunostaining is described herein.

This disclosure further provides a system, method and apparatus forusing single-analyte arrays with a fluidic sample. A single-analytearray can comprise a plurality of wells or micro-wells configured sothat not more than one analyte in a fluid sample will occupy aparticular well. In some aspects, the micro-wells can be micro-cavities.A single-analyte array can also comprise a plurality of patches ormicro-patches configured so that not more than one analyte in a fluidsample will occupy a particular patch. A fluidic sample comprisinganalytes can be introduced into the array and the analytes (e.g., cells)and can be partitioned so that at least 80% of the micro-wells ormicro-patches of the device contain no more than one analyte. In someaspects, the single-analyte array can be used with a microfluidicsystem. In some aspects, the micro-fluidic system can be an eDAR device.

The single-analyte array, in some aspects, involves a method comprisingthe steps of; containment or physical trapping of single cells as thecells are transported in a liquid phase, and following the flow path ofthis phase, transiting to a physically defined position, and residing inthe defined position due to the ensuing flow based forces or surfaceadhesive forces. In another case, the cells are trapped sequentially asthe fluidic flow path is serial with respect of inlet to outlet. Inanother case, multiple fluid flow paths and commensurate multiple singlecells are trapped/sequestered in a parallel manner, due to the numerousflow paths that can simultaneously be experienced by the cells betweenthe inlet and outlet.

The methods, systems, apparatuses and devices described in the presentdisclosure can be used in a wide variety of applications in biology andpathology for the separation, concentration, and/or isolation ofanalytes (e.g., rare cells). For example, some applications can include,but are not limited to, the capture of rare cells (e.g., cancer cells,cancer stem cells, malaria infected erythrocytes, stem cells, fetalcells, immune cells, infected cells) from fluids (e.g., body fluids) fordiagnosis and prognosis of disease; isolation of single-celled parasites(e.g., giardia, cryptosporidium) for water quality monitoring; isolationof infected cells (e.g., lymphocytes, leukocytes) for monitoring diseaseprogression (e.g., HIV, AIDS, cancer); fetal cells in maternal blood forscreening (e.g., disease, genetic abnormalities); stem cells for use intherapeutic applications; prion-infected cells for prion-related (e.g.,mad cow) disease screening; and others.

In a particular aspect, the rare particle is a rare cell. Rare cells canbe nucleated or non-nucleated. Rare cells include, but are not limitedto, cells expressing a malignant phenotype; fetal cells, such as fetalcells in maternal peripheral blood, tumor cells, such as tumor cellswhich have been shed from tumor into blood or other bodily fluids; cellsinfected with a virus, such as cells infected by HIV, cells transfectedwith a gene of interest; and aberrant subtypes of T-cells or B-cellspresent in the peripheral blood of subjects afflicted with autoreactivedisorders.

As used herein, an “ensemble-decision” refers to a decision made basedon the detection of the presence or absence of a characteristic in anensemble, or a group, of particles. A group can comprise at least 3particles, analytes and/or cells. In some aspects, an ensemble-decisionwill be made based on the presence or absence of a single distinctparticle in an aliquot of a fluid sample containing a plurality ofparticles. Importantly, ensemble-decisions made based on the presence orabsence of a single particle will be applied to the entire aliquot(i.e., to all of the particles present in the aliquot).

As used herein, an “aliquot” refers to a portion of the total volume ofa fluid sample to be analyzed. An aliquot can contain at least oneparticle, analyte or cell. An aliquot can contain a group of particles,analytes or cells. An aliquot occupies a three-dimensional space and theparticles within distribute randomly without organization. An aliquothas a finite depth, and particles can distribute along the depth with nodiscernible layers. In the context of the present application, analiquot is analyzed in its entirety without sub-division.

As used herein, the phrase “partitioning a fluid” refers to separatingor otherwise redirecting a portion or aliquot of a fluid from the totalvolume of a fluid sample.

In certain aspects, an aliquot can consist of a fraction of a largerfluid sample, for example, about ½ of a fluid sample, or about ⅓, ¼, ⅕,⅙, 1/7, ⅛, 1/9, 1/10, or less of a fluid sample. In certain aspects, analiquot can consist of, for example, about 10% of a fluid sample, orabout 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%,0.001%, or less of a fluid sample. As such, a fluid that is to beexamined or processed by an eDAR methodology provided herein can bedivided, for example, into at least about 2 aliquots, or at least about3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200,225, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100,1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500,3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000,20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000,200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000,1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7million, 8 million, 9 million, 10 million, or more aliquots. One ofskill in the art would understand that the number of aliquots into whicha fluid sample would be partitioned into will depend upon the number ofrare particles expected in the fluid and the total volume of the fluidsample.

In certain aspects, an aliquot can comprise a fraction of a larger fluidsample, for example, ½ of a fluid sample, or ⅓, ¼, ⅕, ⅙, 1/7, ⅛, 1/9,1/10, or less of a fluid sample. In certain aspects, an aliquot cancomprise, for example, 10% of a fluid sample, or 9%, 8%, 7%, 6%, 5%, 4%,3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.001%, or less of a fluid sample.As such, a fluid that is to be examined or processed by an eDARmethodology provided herein can be divided, for example, into at least 2aliquots, or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, 600, 700,800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800,1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000,8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000,80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000,700,000, 800,000, 900,000, 1 million, 2 million, 3 million, 4 million, 5million, 6 million, 7 million, 8 million, 9 million, 10 million, or morealiquots. One of skill in the art would understand that the number ofaliquots into which a fluid sample would be partitioned into will dependupon the number of rare particles expected in the fluid and the totalvolume of the fluid sample.

In certain aspects, an aliquot can have a volume, for example, ofbetween about 0.1 nL and about 10 mL, or between about 1 nL and about 1mL, or between about 1 nL and about 100 μL, or between about 1 nL andabout 10 μL, or between about 1 nL and about 1 μL, or between about 1 nLand about 100 nL, or between about 0.1 nL and about 10 nL.

In certain aspects, an aliquot can have a volume, for example, ofbetween 0.1 nL and 10 mL, or between 1 nL and 1 mL, or between 1 nL and100 μL, or between 1 nL and 10 μL, or between 1 nL and 1 μL, or between1 nL and 100 nL, or between 0.1 nL and 10 nL.

As used herein, the term “ranking” refers to assessing a quantitativeproperty, qualitative property, or importance of an aliquot bycategorization. In one aspect, an aliquot can be ranked as either null(for example, when a rare particle is not detected in the aliquot) ornonzero (for example, when at least one rare particle is detected in analiquot). In one aspect, the ranking can be binary. In other aspects, analiquot can be ranked according to additional categories, for example,which correlate with the concentration of the rare particle in thealiquot, the identity of the rare particle in the aliquot, theidentities of a plurality of different rare particles in the aliquot,and the like. In this fashion, any number of categories can be assignedbased on ranges of concentration, for example, between about 1 and 10,between about 11 and 20, between about 1 and 50, between about 51 and100, between about 1 and 100, between about 101 and 201, etc. In someaspects, the number of categories can be assigned based on ranges ofconcentration, for example, between 1 and 10, between 11 and 20, between1 and 50, between 51 and 100, between 1 and 100, between 101 and 201,etc. These rankings can be assigned an arbitrary number corresponding toone of a number of predetermined quantitative or qualitative categories(e.g., 0, 1, 2, 3, 4, 5, etc.), or a number corresponding to an actualvalue for the number or approximate number or rare particles in thealiquot.

As used herein, the term “microfluidic chip” is used interchangeablywith the terms chip, fluidic chip, microchip or fluidic microchip.

As used herein, a “computer” refers to at least a digital processor. Thedigital processor can be, but is not limited to, a field programmablegate array (FGPA), application specific integrated circuit (ASIC) orreal-time (RT) processor.

Devices, Apparatuses and Methods for Performing Assays

The methods described in the present disclosure can be used for theisolation and detection of an analyte from a fluid sample. In someaspects, the method can include detection of molecules on the analyteusing a first tag or a first set of tags, reduction of the signalemitted by the tags and detection of another set of molecules on theanalyte using a second tag or second set of tags. In some aspects, themethod can be performed using analytes separated from a sample. In someaspects, the method can be combined with a microfluidic device. Themicrofluidic device can be used to isolate analytes from a sample. Insome aspects, the method, referred to as immunostaining and bleaching,can be performed on the microfluidic device used to isolate analytesfrom a sample.

A concept diagram illustrating an exemplary case of the immunostainingand photobleaching method is shown in FIG. 1. The method ofimmunostaining and photobleaching can be performed on the microfluidicdevice 100 along with a detection scheme 155. The microfluidic device100 can be placed on a stage 160 of a microscope above or under a sourceof light 180 and a source of radiation 175. The analyte can beintroduced to the microfluidic device 100 and eDAR can be performed 110to isolate the analyte from the fluid sample. The isolated analyte canbe directed to the staging chamber 115 where the analyte can remain forthe duration of the immunostaining and photobleaching method. Theanalyte can be contacted with one or more tags 120. A source ofradiation 175 can radiate the one or more tags on the analyte 125 andthe signal emitted by the one or more tags can be detected 130. Thesource of radiation 175 can originate from a laser, or light emittingdiode (LED), or a lamp 190. The signal emitted by the tag can be reduced135 using a source of light 180. During the reduction of the signal 135,a digital processor (e.g., computer) and software 165 and acharge-coupled device (CCD) 170 can be used to detect 130 the durationof the signal emitted by the tag. In some aspects, the exposure time ofthe tagged analyte to the source of illumination can persist until thesignal emitted by the tag is eliminated. Steps 120, 125, 130 and 135comprise one cycle of immunostaining and photobleaching. The cycles 140of immunostaining and photobleaching can be repeated until multiplecycles are complete. The final round of photobleaching 135 may or maynot be performed during the final cycle. After detection of the analyte130, the final cycle can proceed to analyze the signals emitted by thetags 150.

A concept diagram illustrating an exemplary case of the eDAR apparatus200 that can be used in combination with the immunostaining andphotobleaching method 290 is shown in FIG. 2. The fluid sample reservoir225 and channel 230 can connect to the fluid sample inlet 245. Thebuffer reservoir 215 can connect to a source of pressurized gas 205through a line 235. The buffer inlet 250 can be connected to the fluidsample inlet 245. The filtration area 270 can be connected to the bufferinlet 250, the fluid sample inlet 245, the outlet 275 and the wastechamber 280. A fluid sample can enter the eDAR apparatus 200 and ananalyte or a plurality of analytes can be trapped on the filtrationstage 270. The filtration stage can contain a plurality of chamberswhere each chamber can contain a single analyte. The immunostaining andphotobleaching method 290 can be performed on a section of themicrofluidic chip 260 which can contain the filtration stage 270.

This disclosure provides a method for sequential immunostaining andphotobleaching that can be performed using a single-analyte array. Thesingle-analyte array allows for sequestration, trapping, manipulation,and detection of single-analytes (e.g., cells). Often, the cells aretrapped by forces generated from fluid flow, gravity, surface adhesiveforces, chemical forces, or optical forces. In some aspects, thesingle-analyte array wells are functionalized with an element that canbind (e.g., covalently, ionically, etc.) the trapped cell. Trapped cellscan be contacted with a chemical agent. The chemical agent can be alabel used to detect exterior molecules, or penetrate the cell membraneand label intracellular molecules. Labeled cells can be imaged andfurther analyzed.

A concept diagram illustrating an exemplary case of the single-analytearray in combination with an immunostaining method is shown in FIG. 3.The fluidic single-analyte array 315 can be part of a microfluidic chipor structure and placed on a microfluidic stage 310. The method ofimmunostaining can be performed on the fluidic single-analyte array 315along with a detection scheme 350. The microfluidic chip stage 310 canbe placed on a stage 355 of a microscope above or under a source oflight 370 and a source of radiation 375. The analytes in a fluid samplecan be introduced to the fluidic single-analyte array 300. The isolatedanalytes can be directed to an area of the fluidic single-analyte array320 where the trapped single-analytes can remain for the duration of theimmunostaining method. The analytes can be prepared 325 using a varietyof methods which can include, but are not limited to; permeabilizationand/or fixation. The analyte can be contacted with one or more tags 330.A source of radiation 375 can radiate 335 the one or more tags on theanalyte and the signal emitted by the one or more tags can be detected340. The source of radiation 375 can originate from a laser 380. Theanalytes can undergo further processing and analysis 345. A digitalprocessor (e.g., computer) and software 360, and a charge-coupled device(CCD) 365 can be used to detect the signal emitted by the tag.

For sequential immunostaining and photobleaching, the signal emitted bythe tag can be reduced using a source of light 370. During the reductionof the signal, a computer and software 360, and a charge-coupled device(CCD) 365 can be used to detect the intensity of the signal emitted bythe tag. The exposure time of the tagged analytes to the source ofillumination can persist until the signal emitted by the tag iseliminated. Steps 330, 335, 340 and 345 comprise one cycle ofimmunostaining and photobleaching. The cycles can be repeated untilmultiple cycles are complete.

The eDAR apparatus can be used for the identification and isolation ofanalytes (e.g., rare cells). eDAR can process a sample in aliquots andcan collect rare cells in a channel or a chamber by an active sortingstep controlled by a hydrodynamic switching scheme. An “all-in-onemicrofluidic chip,” referred to herein as microfluidic chip, withchannels and chambers can be used for sorting rare cells. Themicrofluidic chip can be composed, in part, of a functional area, amicrofabricated filter. eDAR can be used to rapidly (e.g., less than orequal to 12.5 minutes per 1 mL) analyze a fluid containing a mixedpopulation of analytes (e.g., whole blood at greater than or equal toone milliliter) with a high recovery ratio (e.g., greater than or equalto 90%) and a low false positive rate (e.g., close to zero).

The general structure of the microfluidic chip and an exampleconfiguration of eDAR is depicted in FIG. 7A. The bottom left channelcan be used to collect sorted aliquots and can be used to transfer themto the subsequent purification (e.g., purification chamber) area (e.g.,20,000 microslits). The area marked with a dashed box is furtherdepicted in FIG. 7B-D. An example flow condition when no positivealiquot was ranked is shown in FIG. 7B. The blood flow can be switchedto the collection channel, and the sorted aliquot can be confirmed bythe second window in FIG. 7C. FIG. 7D shows that the blood flow can beswitched back after sorting the aliquot.

The apparatus can have several flow rates. The flow rates can refer tothe rate in which a fluid flows through the eDAR apparatus and anycomponents attached to the apparatus. In some aspects, exemplary flowrates can be less than about 5 μL/min, 10 μL/min, 20 μL/min, 25 μL/min,30 μL/min, 35 μL/min, 40 μL/min, 41 μL/min, 42 μL/min, 43 L/min, 44μL/min, 45 μL/min, 46 μL/min, 47 μL/min, 48 μL/min, 49 μL/min, 50μL/min, 51 μL/min, 52 μL/min, 53 μL/min, 54 μL/min, 55 μL/min, 56μL/min, 57 μL/min, 58 μL/min, 59 μL/min, 60 μL/min, 61 μL/min, 62μL/min, 63 μL/min, 64 μL/min, 65 μL/min, 66 μL/min, 67 μL/min, 68μL/min, 69 μL/min, 70 μL/min, 71 μL/min, 72 μL/min, 73 μL/min, 74μL/min, 75 μL/min, 76 μL/min, 77 μL/min, 78 μL/min, 79 μL/min, 80μL/min, 81 μL/min, 82 μL/min, 83 μL/min, 84 μL/min, 85 μL/min, 86μL/min, 87 μL/min, 88 μL/min, 89 μL/min, 90 μL/min, 91 μL/min, 92μL/min, 93 μL/min, 94 μL/min, 95 μL/min, 96 μL/min, 97 μL/min, 98μL/min, 99 μL/min, 100 μL/min, 105 μL/min, 110 μL/min, 115 μL/min, 120μL/min, 125 μL/min, 130 μL/min, 140 μL/min, 150 μL/min, 160 μL/min, 170μL/min, 180 μL/min, 190 μL/min, 200 μL/min, 225 μL/min, 250 μL/min, 275μL/min, 300 μL/min, 350 μL/min, 400 μL/min, 450 μL/min, 500 μL/min, 600μL/min, 700 μL/min, 800 μL/min, 900 μL/min or 1000 μL/min.

In some aspects, the flow rate can be within the range of about 5μL/min-30 μL/min, 15 μL/min-50 μL/min, 25 μL/min-75 μL/min, 40 μL/min-80μL/min, 50 μL/min-90 μL/min, 60 μL/min-100 μL/min, 800 μL/min-160μL/min, 90 μL/min-180 μL/min, 100 μL/min-200 μL/min, 150 μL/min-300μL/min, 200 μL/min-400 μL/min, 300 μL/min-500 μL/min, 400 μL/min-600μL/min, 500 μL/min-700 μL/min, 600 μL/min-800 μL/min, 700 μL/min-900μL/min or 800 μL/min-1000 μL/min.

In some aspects, exemplary flow rates can be less than 5 μL/min, 10μL/min, 20 μL/min, 25 μL/min, 30 μL/min, 35 μL/min, 40 μL/min, 41μL/min, 42 μL/min, 43 L/min, 44 μL/min, 45 μL/min, 46 μL/min, 47 μL/min,48 μL/min, 49 μL/min, 50 μL/min, 51 μL/min, 52 μL/min, 53 μL/min, 54μL/min, 55 μL/min, 56 μL/min, 57 μL/min, 58 μL/min, 59 μL/min, 60μL/min, 61 μL/min, 62 μL/min, 63 μL/min, 64 μL/min, 65 μL/min, 66μL/min, 67 μL/min, 68 μL/min, 69 μL/min, 70 μL/min, 71 μL/min, 72μL/min, 73 μL/min, 74 μL/min, 75 μL/min, 76 μL/min, 77 μL/min, 78μL/min, 79 μL/min, 80 μL/min, 81 μL/min, 82 μL/min, 83 μL/min, 84μL/min, 85 μL/min, 86 μL/min, 87 μL/min, 88 μL/min, 89 μL/min, 90μL/min, 91 μL/min, 92 μL/min, 93 μL/min, 94 μL/min, 95 μL/min, 96μL/min, 97 μL/min, 98 μL/min, 99 μL/min, 100 μL/min, 105 μL/min, 110μL/min, 115 μL/min, 120 μL/min, 125 μL/min, 130 μL/min, 140 μL/min, 150μL/min, 160 μL/min, 170 μL/min, 180 μL/min, 190 μL/min, 200 μL/min, 225μL/min, 250 μL/min, 275 μL/min, 300 μL/min, 350 μL/min, 400 μL/min, 450μL/min, 500 μL/min, 600 μL/min, 700 μL/min, 800 μL/min, 900 μL/min or1000 μL/min.

In some aspects, the flow rate can be within the range of 5 μL/min-30μL/min, 15 μL/min-50 μL/min, 25 μL/min-75 μL/min, 40 μL/min-80 μL/min,50 μL/min-90 μL/min, 60 μL/min-100 μL/min, 800 μL/min-160 μL/min, 90μL/min-180 μL/min, 100 μL/min-200 μL/min, 150 μL/min-300 μL/min, 200μL/min-400 μL/min, 300 μL/min-500 μL/min, 400 μL/min-600 μL/min, 500μL/min-700 μL/min, 600 μL/min-800 μL/min, 700 μL/min-900 μL/min or 800μL/min-1000 μL/min.

The apparatus can have several sorting efficiencies. The sortingefficiency can refer to the recovery of analytes of interest. In someaspects, an exemplary sorting efficiency can be greater than about 5%,10%, 20%, 25%, 30%, 35%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some aspects,the sorting efficiency can be within the range of about 5%-30%, 15%-50%,25%-75%, 40%-80%, 50%-90% or 60%-100%.

In some aspects, an exemplary sorting efficiency can be greater than 5%,10%, 20%, 25%, 30%, 35%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some aspects,the sorting efficiency can be within the range of 5%-30%, 15%-50%,25%-75%, 40%-80%, 50%-90% or 60%-100%.

The eDAR apparatus can have several recovery ratios. The recovery ratiocan refer to the recovery of analytes of interest. In some aspects, therecovery ratio can be greater than about 5%, 10%, 20%, 25%, 30%, 35%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100%. In some aspects, the recovery ratio can bewithin the range of about 5%-30%, 15%-50%, 25%-75%, 40%-80%, 50%-90% or60%-100%.

In some aspects, the recovery ratio can be greater than 5%, 10%, 20%,25%, 30%, 35%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In some aspects, the recovery ratio can be within the range of 5%-30%,15%-50%, 25%-75%, 40%-80%, 50%-90% or 60%-100%.

In some aspects an eDAR apparatus or method is used to separate a firstcell type from a second cell type. In some aspects, an isolated samplecomprises greater than 5%, 10%, 20%, 25%, 30%, 35%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% of a total number of the first cell type. In some aspects, anisolated sample comprises less than than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%,35%, 40%, 45, 50%, 60%, 70%, 80%, or 90% of a total number of the secondcell type.

The eDAR apparatus can include a microscope equipped with sources ofradiation (e.g., lasers) for excitation and a mode of detection, sourcesof light (e.g., photobleaching), a timer, tubing, a waste-collectiondevice, a camera for imaging tagged analytes (e.g., cells), pumps tocontrol the flow of fluid in and out of the microfluidic chip, a digitalprocessor to control the active sorting step and a digital processor(e.g., computer system) for processing images. The digital processor canbe a computer.

In some aspects, the eDAR platform can include an apparatus for thecapture of more than one analyte (e.g., rare cell). The “dual-capture”eDAR can separate multiple rare cells from a mixed sample. The mixedsample (e.g., fluid sample) can be labeled with a detection reagent andentered into the top of the “dual-capture” apparatus at a main channel.Two side channels can be used to control the hydrodynamic switching ofthe flow of the mixed sample. In some aspects, the flow can becontrolled using two solenoids. Two subpopulations of rare cells can beseparated and trapped on two different filtration areas on the samemicrofluidic chip.

In various aspects, apparatuses are provided for partitioning cellsexpressing a specific biomarker profile from a sample derived from afluid, wherein: the apparatuses comprise a set of tubing connected to amicrofluidic chip that has at least one channel and a chamber; and theapparatuses are capable of isolating the cells in the chamber, wherein,after isolation, the chamber comprises greater than 80% of the totalpopulation of cells in the sample expressing the specific biomarkerprofile and wherein, after isolation, the chamber comprises less than 5%of the total population of cells in the sample expressing a differentbiomarker profile.

In some aspects, the isolation of the cells expressing a specificbiomarker profile occurs in less than 20 minutes. In some aspects, thespecific biomarker profile is present on less than 5% of the cells inthe sample of fluid. In certain aspects, the fluid is blood. In furtheraspects, the fluid is fractionated whole blood. In still furtheraspects, the fluid is the nucleated cell fraction of whole blood.

In various aspects, systems are provided for detecting a particle in afluid sample, the systems comprising: a microfluidic chip comprising aninput channel, a first output channel, a second output channel, and adirectional flow channel; a valve, wherein the valve is separable fromthe microfluidic chip, and wherein: the valve regulates the flow of afirst fluid in the directional flow channel; and the flow of the firstfluid in the directional flow channel directs the flow of a second fluidfrom the input channel to the first output channel, the second outputchannel, or a combination thereof; a detector configured to detect asignal emitted from a portion of the second fluid in the input channel;and a processor configured to: assign a value to the portion based onthe signal; and operate the valve. In some aspects, the valve is anelectro-actuated valve.

In various aspects, systems are provided for detecting a particle in afluid sample, the system comprising: (a) a microfluidic chip comprisingat least one sample input channel, at least one directional flowchannel, and at least two output channels, wherein the at least onedirectional flow channel intersects the sample input channel; (b) anelectro-actuated valve that is located on a device that is not part ofthe microfluidic chip, wherein the electro-actuated valve controls theflow of a fluid by controlling an input channel that intersects at leastone directional flow channel or at least one of the at least two outputchannels; (c) at least one detector capable of detecting one or moreanalytes in an aliquot of the fluid sample; and (d) a processor capableof assigning a value to the aliquot based on the presence, absence,identity, composition, or quantity of analytes in the aliquot, whereinthe processor is in communication with the detector and theelectro-actuated valve.

In some aspects, the electro-actuated valve is a solenoid valve. Incertain aspects, the electro-actuated valve controls the flow of thefluid in at least one directional flow channel. In further aspects, theelectro-actuated valve is normally closed and wherein theelectro-actuated valve opens after receiving a signal from theprocessor. In still further aspects, the electro-actuated valve isnormally open and wherein the electro-actuated valve closes afterreceiving a signal from the processor.

In some aspects, at least one directional flow channel comprises atleast two ports and wherein the electro-actuated valve controls the flowof fluid through one of the ports. In certain aspects, theelectro-actuated valve directly controls the flow of a fluid in at leastone directional flow channel. In some aspects, the electro-actuatedvalve directly controls the flow of a fluid in a channel that feeds intoat least one directional flow channel. In certain aspects, theelectro-actuated valve directly controls the flow of a fluid in only onedirectional flow channel. In further aspects, the electro-actuated valvedirectly controls the flow of a fluid in an output channel. In stillfurther aspects, the electro-actuated valve directly controls the flowof a fluid in a channel that feeds into an output channel. In someaspects, the electro-actuated valve is a piezo-electric valve.

In some aspects, a directional flow channel intersects an outputchannels at a junction. In certain aspects, the detector is located onat least one channel that is not an output channel. In some aspects, thedevice comprises a confirmatory laser. In certain aspects, theconfirmatory laser is located on at least one channel that is not aninput channel. In further aspects, the system comprises a seconddetector. In still further aspects, at least one channel is in fluidiccommunication with a filter.

In various aspects, devices are provided for detecting a rare particlein a fluid sample, the devices comprising: an input channel; a firstoutput channel; a second output channel; a detector configured to detectthe presence or absence of a particle in a portion of the fluid sample;a mechanism for directing the flow of the portion from the input channelto the first output channel, the second output channel, or a combinationthereof based on the presence or absence of the particle; and a filterin fluidic communication with the first output channel. In some aspects,the filter comprises an array of apertures. In certain aspects, asmallest dimension of each aperture in the array is smaller than asmallest dimension of the particle.

In various aspects, devices are provided for detecting a rare particlein a fluid sample, the devices comprising: (a) at least one sample inputchannel; (b) at least two output channels, wherein at least one of thetwo output channels is in fluidic communication with an array ofapertures; (c) at least one detector capable of detecting one or morerare particles in an aliquot of the fluid sample; and (d) a mechanismfor sorting the one or more rare particles by directing the flow ofaliquots containing the one or more rare particles through a firstoutput channel.

In some aspects, the device comprises a first output channel and asecond output channel. In certain aspects, the mechanism directs theflow of the aliquots into the second output channel if the aliquot doesnot contain a rare particle. In certain aspects, the mechanism forsorting comprises an electrode, a magnetic element, an acoustic element,or an electro-actuated element.

In some aspects, the array of apertures is disposed between an inputchannel and an output channel. In certain aspects, the array ofapertures is in the same plane as an input channel and an outputchannels. In further aspects, the array of apertures is disposed withinthe first output channel. In still further aspects, the array ofapertures is disposed in a chamber in fluidic communication with thefirst output channel. In some aspects, the array of apertures isconfigured so that the rare particles cannot pass through the aperturesbut at least one other particle is capable of passing through theapertures. In certain aspects, the array of apertures is configured sothat the rare particles are capable of passing through the apertures butat least one other particle cannot pass through the apertures. Infurther aspects, the array of apertures comprises greater than 1000apertures.

In certain aspects, the detector is selected from the group consistingof a camera, an electron multiplier, a charge-coupled device (CCD) imagesensor, a photomultiplier tube (PMT), an avalanche photodiode (APD), asingle-photon avalanche diode (SPAD), a silicon photomultiplier (SiPM),and a complementary metal oxide semiconductor (CMOS) image sensor.

In various aspects, integrated systems are provided for performing anassay, the systems comprising: a fluid sample comprising a particle,wherein the particle comprises a marker; an input channel; a firstoutput channel; a second output channel; a first detector configured todetect the presence or absence of the particle in a portion of the fluidsample, the portion disposed within the input channel; a mechanism fordirecting the flow of the portion from the input channel to the firstoutput channel based on the presence or absence of the particle; amicro-cavity in fluidic communication with the first output channel andconfigured to trap the particle; and a second detector configured todetect the presence or absence of the marker in the micro-cavity.

Microfluidic Chip Design and Fabrication

The microfluidic chip can be fabricated to provide for an efficientactive sorting scheme and subsequent purification (e.g., purificationchamber) scheme. The microfluidic chip can be composed of two layers ona silicon master and can be fabricated with one-step replica moldinginto polydimethylsiloxane (PDMS). The microfluidic chip can be finishedwith bonding to a glass substrate.

In some aspects, the silicon master can be fabricated using twophotolithography processes (FIG. 5). The features can be designed usingstandard software (e.g., AutoCAD, Autodesk, San Rafael, Calif.), and canbe written on a chrome mask. In these cases, positive resist lithographyand deep reactive ion etching (DRIE) can be used to form the first layer(FIG. 5). The first layer can be the micro-filter feature. In someaspects, the positive photo resist (e.g., AZ 1512) is achieved by aprocess that can include a DRIE process. The DRIE process can achieve adepth (e.g., 4.5-5 μm) suitable for various features.

In some aspects, the second layer of the eDAR microfluidic chip featurescan be fabricated using a negative photoresist (e.g., SU-8-3050 fromMicroChem, Newton, Mass.), and the height of the feature can becontrolled (e.g., 50 μm). The master can be silanized using, forexample, tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane(Sigma-Aldrich, St. Louis, Mo.). The silanized master and silicon wafercan be coated with uncured PDMS and baked (e.g., for 2 hours at 70° C.).In some aspects, the piece of PDMS with the desired micro-features canbe peeled off the silicon master, and then bonded with a piece of coverglass using the standard process of plasma oxidation to complete thefabrication of the eDAR microfluidic chip.

The microfluidic chip can contain specialized regions including, astaining region, a photobleaching region, an imaging region, andadditional regions. In some aspects, the regions can be the same regionused for at least one purpose. In other cases, the regions can bedifferent regions use for one purpose. In other cases, the regions canbe different regions use for at least one purpose. Each region can beused for more than one purpose. In some aspects, the eDAR microfluidicchip includes an integrated filtration area fabricated by standardlithography methods. In some aspects, the microfluidic chip can have twointegrated functional areas, the eDAR sorting area and a slit-structurebased filtration unit.

Channels in the Microfluidic Chip

The disclosure provided herein describes an apparatus for detecting ananalyte (e.g., rare particle) in a fluid sample, the apparatuscomprises: (a) at least a first input channel; (b) at least two exitchannels; (c) at least one detector capable of detecting one or morerare particles in an aliquot of the biological fluid; (d) a mechanismfor directing the flow of the aliquot; and (e) a ranking device capableof assigning a value to the aliquot based on the presence, absence,identity, composition, or quantity of the rare particles in the aliquot,wherein the digital processor (e.g., computer) is in communication withthe detector and the mechanism for directing the flow of the aliquot.

In some aspects, the apparatus provided herein can comprise a flowchannel enclosed by walls and/or microfabricated on a substrate, withdesign features to minimize inadvertent damage to analytes (e.g., rarecells). Reducing inadvertent damage of rare cells can reduce the rate offalse-negatives that cause erroneous patient diagnosis or prognosis. Theflow channel can further comprise channels with hydrodynamicallydesigned apertures to exclude biological cells with minimal stress ordamage. The flow channel is as described in US Patent Application Nos.2007/0037172 and 2008/0248499. Such channels, referred to in theaforementioned patent applications as channels with one-dimensional(“1-D”) apertures, reduce the hydrodynamic pressure experienced by thecells during the cell exclusion process and therefore reduce thelikelihood of cell lysis. Channels with 1-D apertures can bestrategically arranged in an array according to “effusive filtration”configuration as described in US Patent No. 2008/0318324 to furtherre-direct, partition, dampen, or disperse the flow, consequentlyreducing the force of impact experienced by the cells at the moment ofexclusion. The walls that enclose the flow channel can be fabricatedusing a UV-curing process in accordance with the procedures described inPCTPCT/US2009/02426, from a biocompatible substrate material that is amedical-device grade polymer, so that the eDAR apparatus can be incompliance with regulations governing medical device manufacturing.

The main channel in the sorting area, which can be used to introducefluid into the sorting junction, can have a particular height (e.g., 50μm) and a particular width (e.g., 150 μm). In most cases, the otherchannels (e.g., four) can have a particular height (e.g., 50 μm) and aparticular width (e.g., 200 μm). The slit-structure based filters in thefiltration unit can have a particular height (e.g., 5 μm) and aparticular width (e.g., 5 μm). The microfluidic chip can contain up toand can include 50,000 slits in the slit structure.

In some aspects, the eDAR apparatus can further comprise channels forchanneling said aliquots based on said ranking. The channels can betreated with anticoagulant compounds, compounds that preferentially bindto the analytes (e.g., rare bioparticles), compounds that preventagglomeration of rare bioparticles or their combinations.

In some aspects, the eDAR apparatus provided herein can comprise aplurality of flow channels, including one or more input flow channels(e.g., channels that bring an aliquot to a detection volume) and one ormore output channels (e.g., channels that take an aliquot away from adetection volume. In some aspects, the eDAR apparatus as provided hereincan comprise a combination of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, or more input channels and at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, or more output channels. In someaspects, the eDAR apparatus as provided herein can comprise acombination of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, ormore input channels and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, or more output channels. In some aspects, an apparatus can comprisemultiple flow channels connecting to the main channel to injectadditional fluid to alter the local velocity.

In some aspects, a fluid is delivered from a channel that is part of amicrofluidic chip to a chamber that is in fluidic communication with thechannel but is external to the microfluidic chip. In some aspects, thechamber is a vial. In other aspects, the chamber is a single well or awell in a well plate. In further aspects, the chamber is amicrocentrifuge tube. In further aspects, the chamber is amicrocentrifuge tube, wherein the microcentrifuge tube is an Eppendorftube. Any suitable structure can be used for the vial and one ofordinary skill in the art could readily identify suitable chambers foruse with the present disclosure.

In some aspects, a fluid is delivered from a channel to a chamber thatis in fluidic communication with the channel via a tube or othersuitable structure for transporting a fluid. The tube can comprise amaterial constructed from a biocompatible polymer. In other aspects, thechamber can be in fluidic communication with the channel via a capillarytube, such as a fused silica capillary tube, such as is used e.g., forperforming capillary electrophoresis. Other types of tubing suitable forbringing a channel into fluidic communication with a chamber will bereadily apparent to one of ordinary skill in the art.

As used herein, the term “in fluidic communication with” (and variationsthereof) refers to the existence of a fluid path between components.Being in fluidic communication neither implies nor excludes theexistence of any intermediate structures or components. Two componentscan be in fluidic communication even if in some instances the pathbetween them is blocked and/or fluid is not flowing between them. Thus,intermittent fluid flow is contemplated in certain aspects where thereis fluidic communication.

In some aspects, an apparatus provided herein can comprise a flowchannel or chamber enclosed by walls fabricated from materialsincluding, but not limited to, polymeric materials (polydimethylsiloxane(PDMS), polyurethane-methacrylate (PUMA), polymethylmethacrylate (PMMA),polyethylene, polyester (PET), polytetrafluoroethylene (PTFE),polycarbonate, parylene, polyvinyl chloride, fluoroethylpropylene,lexan, polystyrene, cyclic olefin copolymers, polyurethane,polyestercarbonate, polypropylene, polybutylene, polyacrylate,polycaprolactone, polyketone, polyphthalamide, cellulose acetate,polyacrylonitrile, polysulfone, epoxy polymers, thermoplastics,fluoropolymer, and polyvinylidene fluoride, polyamide, polyimide),inorganic materials (glass, quartz, silicon, GaAs, silicon nitride),fused silica, ceramic, glass (organic), metals and/or other materialsand combinations thereof.

In some aspects, wall materials can be fabricated of porous membranes,woven or non-woven fibers (such as cloth or mesh) of wool, metal (e.g.,stainless steel or Monel), glass, paper, or synthetic (e.g., nylon,polypropylene, polycarbonate, parylene, and various polyesters),sintered stainless steel and other metals, and porous inorganicmaterials such as alumina, silica or carbon.

In some aspects, the apparatus provided herein can comprise a flowchannel or chamber that has been pre-treated with a chemical orbiological molecule. For example, a channel or chamber can be treatedwith an anticoagulant compound to prevent or reduce the association ofan analyte (e.g., rare particle or cell) in the fluid sample, a compoundthat preferentially binds to an analyte (e.g., rare particle or cell),or a compound that prevents or reduces the agglomeration or aggregationof a analyte (e.g., rare particle or cell) in the fluid sample.

In some aspects, the channel or chamber surfaces can be treated withanticoagulant compounds, compounds that preferentially bind tocirculating tumor cells, or compounds that prevent the sticking ofcells.

In some aspects, a channel or chamber surface can be modified chemicallyto enhance wetting or to assist in the adsorption of select cells,particles, or molecules. Surface-modification chemicals can include butnot limited to silanes such as trimethylchlorosilane (TMCS),hexamethyldisilazane (HMDS),(Tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane,chlorodimethyloctylsilane, Octadecyltrichlorosilane (OTS) orγ-methyacryloxypropyltrimethyoxy-silane; polymers such as acrylic acid,acrylamide, dimethylacrylamide (DMA), 2-hydroxyethyl acrylate,polyvinylalcohol (PVA), poly(vinylpyrrolidone (PVP), poly(ethyleneimine) (PEI), Polyethylene glycol (PEG), epoxy poly(dimethylacrylamide(EPDMA), or PEG-monomethoxyl acrylate; surfactants such as Pluronicsurfactants, Poly(ethylene glycol)-based (PEG) surfactants, sodiumdodecylsulfate (SDS) dodecyltrimethylammonium chloride (DTAC),cetyltriethylammonium bromide (CTAB), or Polybrene (PB); cellulosederivatives such as hydroxypropylcellulose (HPC), orhydroxypropylmethylcellulose (HPMC); amines such as ethylamine,diethylamine, triethylamine, or triethanolamine, fluorine-containingcompounds such as those containing polytetrafluoroethylene (PTFE) orTeflon.

Filtration

The eDAR apparatus provided herein can further comprise a filterelement. In some aspects, the filter element can be in the form ofmicro-posts, micro-barriers, micro-impactors, micro-sieves, channelswith apertures smaller than bioparticles, channels with apertures suchthat a bioparticle can be prevented from entering an aperture but fluidcan be allowed to continue to flow around the bioparticle through theaperture (“1-D channels”), microbeads, porous membranes, protrusionsfrom the walls, adhesive coating, woven or non-woven fibers (such ascloth or mesh) of wool, metal (e.g., stainless steel or Monel), glass,paper, or synthetic (e.g., nylon, polypropylene, polycarbonate,parylene, and polyester), sintered stainless steel or other metals, orporous inorganic materials such as alumina, silica.

In some aspects, an array of apertures in the filter element isconfigured so that a particle of interest, such as a rare particle orcell, cannot pass through the apertures of the filter element, while atleast one other particle is capable of passing through the apertures ofthe filter element. In other aspects, an array of apertures in thefilter element is configured so that a particle of interest, such as arare particle or cell, is capable of passing through the apertures ofthe filter element, while at least one other particle is incapable ofpassing through the apertures of the filter element.

In some aspects, the eDAR microfluidic chip can be fabricated withmicroslits (FIG. 10a ). The microslits can be used to capture rare cellswithout retaining any additional non-specific particles (e.g., red bloodcells, RBCs). The size of the microslits can vary. In some aspects, theheight of the microslits can be less than or equal to about 0.1 μm, 0.5μm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 50 μm,75 μm or 100 μm tall. In some aspects, the height of the microslits canbe in the range of about 0.1-5 μm, 1-10 μm, 5-15 μm, 10-30 μm, 15-40 μm,20-50 μm, 30-75 μm and 50-100 μm tall. In some aspects, the width of themicroslits can be less than or equal to about 0.1 μm, 0.5 μm, 1 μm, 5μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 50 μm, 75 μm or 100μm tall. In some aspects, the width of the microslits can be in therange of about 0.1-5 μm, 1-10 μm, 5-15 μm, 10-30 μm, 15-40 μm, 20-50 μm,30-75 μm and 50-100 μm tall. For example, the microslits can have aheight of 5 μm and width of 5 μm (FIG. 10b ).

In some aspects, the height of the microslits can be less than or equalto 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm,40 μm, 50 μm, 75 μm or 100 μm tall. In some aspects, the height of themicroslits can be in the range of 0.1-5 μm, 1-10 μm, 5-15 μm, 10-30 μm,15-40 μm, 20-50 μm, 30-75 μm and 50-100 μm tall. In some aspects, thewidth of the microslits can be less than or equal to 0.1 μm, 0.5 μm, 1μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 50 μm, 75 μmor 100 μm tall. In some aspects, the width of the microslits can be inthe range of 0.1-5 μm, 1-10 μm, 5-15 μm, 10-30 μm, 15-40 μm, 20-50 μm,30-75 μm and 50-100 μm tall.

In some aspects, microfluidic chips can be prepared with 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 5000, 20,000, 30,000, 40,000, or50,000 apertures or microslits. In other aspects, microfluidic chips canbe prepared with greater than 100, greater than 200, greater than 300,greater than 400, greater than 500, greater than 600, greater than 700,greater than 800, greater than 900, greater than 1000, greater than5000, greater than 20,000, greater than 30,000, greater than 40,000, orgreater than 50,000 apertures or microslits. In some aspects, pressurescan be lower for microfluidic chips with more microslits. For example,the eDAR microfluidic chip with 20,000 slits required a pressure of lessthan 4 psi on the two side-buffer channels to balance the hydrodynamicswitching process. In some aspects, the lower pressure across themicrofilter can minimize the stress upon and deformation of the isolatedrare cell.

Microslits can be used as a source of filtration. In some aspects, themicroslits can be fabricated from any material that allows for sortingand does not cause aberrations during imaging (e.g., PDMS) and bondedwith a piece of coverslip for use with imaging systems (FIGS. 10c and10D). In some aspects, the microslits can be microfilters.

In some aspects, use of the microslits for filtration purposes canimprove imaging accuracy and enumeration of trapped cells. In someaspects, the microslits can increase the speed and efficiency of asecond round of labeling on trapped rare cells. For example, two cancercells labeled with anti-EpCAM-PE can be trapped on the microslit (FIG.10e ). The cells can be fixed, permeabilzed, and labeled withanti-Cytokeratin-Alexa488, anti-CD45-Alexa700, anti-Her2-Alexa647 andHoechst.

Hydrodynamic Sorting

Two key factors that contribute to the features and performance of theeDAR platform are, (1) an efficient and active sorting scheme and a (2)subsequent efficient purification (e.g., purification chamber) scheme.The analytical performance of the microfluidic chip and hydrodynamicswitching mechanisms can be optimized for a particular recoveryefficiency (e.g., 95%), a particular false positive rate (e.g., 0) and aparticular throughput (e.g., 4.8 mL of whole blood per hour).

In some aspects, the mechanism for directing the flow directs the flowof an aliquot containing a rare particle into one of a plurality of exitchannels depending on the identity, composition, or quantity of the rareparticle. The mechanism for directing the flow of the aliquot can eitherbe into a first exit channel, if the aliquot contains a rare particle,or a second exit channel, if the aliquot does not contain a rareparticle.

In some aspects, the mechanism for directing the flow of the aliquot cancomprise an electrode, a magnetic element, an acoustic element, anelectro-actuated element, an electric field, a piezo-electric valve, ora magnetic field. In other cases, the mechanism for directing the flowof the aliquot can comprise one or more electro-actuated valves orpistons, wherein the valves or pistons control the flow of a liquid inat least a first directional flow channel that intersects with the firstinput channel and the two exit channels at a first junction.

In some aspects, the apparatus provided herein can comprise one or moreelectrodes for tracking and/or manipulating the trajectory or flow of aparticle, aliquot, or fluid sample. In some aspects, the apparatusprovided herein can comprise one or more electrodes for tracking and/ormanipulating the trajectory or flow of an ensemble, or a group, ofparticles or aliquots. In this case, the electrode can enhance theseparation of an aliquot based on phenomena such dielectrophoresis orelectrowetting.

In other cases, the apparatuses provided herein can further comprise amagnetic element for the separation of a rare particle (e.g., cell)bound to or bound by a magnetic particle. In other cases, theapparatuses provided herein can further comprise a magnetic element forthe separation of an ensemble or group of rare particles (e.g., cells)bound to or bound by at least one magnetic particle. In some aspects,the magnetic element can enhance the separation of an aliquot, particle,or cell based on the magnetic susceptibility of the cells or themicro-magnetic or nano-magnetic particles attached to a particle orcell. In some aspects, the magnetic element can enhance the separationof an ensemble or a group of particles, or cells based on the magneticsusceptibility of the cells or the micro-magnetic or nano-magneticparticles attached to at least one particle or cell.

The eDAR apparatus can have a second line-confocal detection windowlocated on the collection side to monitor the efficiency of thehydrodynamic switching in real time. In some aspects, the eDAR apparatuscan be paired with confocal imaging.

In some aspects, the hydrodynamic switch can be controlled by a solenoidand the pressure drop in the two side buffer lines. A solenoid can belocated in the rare cell collection channel. In some aspects, thissolenoid is in the closed position on the left and the “negative”aliquots flow into the waste channel on the right (FIG. 4A). When thesolenoid is opened, a pressure drop between the two side channels thatcontain buffer switches the blood flow from the waste channel to thecollection side. This switch can occur in less than 10 milliseconds(e.g., 2-3 ms) to collect rare particles (e.g., cells).

In some aspects, various hydrodynamic sorting schemes can be used. Thedisclosure provides for eight different hydrodynamic sorting schemes(FIG. 6). The fluid sample (e.g., blood) can be injected from the mainchannel, shown as the dark black flow. Buffer (light grey color in themicrofluidic chip) can flow in the two side channels, rare cells can becollected to the bottom left channel, and the waste can be directed tothe bottom right channel. The rectangular blocks represent the solenoid(see “solenoids”). The solenoid can be set to be normally open (N.O.) ornormally closed (N.C.), as indicated by a dark grey or light grey block,respectively (FIG. 6).

Channels of the microfluidic chip may intersect at junctions. In someaspects, one channel intersects with a different channel at a junction.In some aspects, one channel intersects with more than one differentchannels at a junction. In some aspects, one channel intersects with 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 differentchannels at a junction. In some aspects, more than one channelintersects with a different channel at a junction. In some aspects, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 differentchannels intersect with one different channel at a junction.

Channels of the microfluidic chip may not intersect at junctions. Insome aspects, one channel intersects with a different channel at alocation on the microfluidic chip that is not a junction. In someaspects, one channel intersects with more than one different channels ata location on the microfluidic chip that is not a junction. In someaspects, one channel intersects with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95 or 100 different channels at a location on themicrofluidic chip that is not a junction. In some aspects, more than onechannel intersects with a different channel at a location on themicrofluidic chip that is not a junction. In some aspects, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 different channelsintersect with one different channel at a location on the microfluidicchip that is not a junction.

Solenoids and Regulation of Flow

The eDAR apparatus includes solenoids to control flow of fluid andhydrodynamic sorting schemes. In some aspects, solenoids can be pistons.For example, solenoid pistons are subcomponents of electro-actuatedsolenoid valves. In some aspects, the electro-actuated solenoid valvescan be piezo-electric valves. In some aspects, solenoid pistons can beembedded in the apparatus by molding. In some aspects, solenoids can bevalves. For example, the embedded solenoid pistons may be replaced bysolenoid valves in fluidic communication via tubings.

In some aspects, the eDAR apparatus can contain one solenoid. In othercases, the eDAR apparatus can contain more than one solenoid. Forexample, the eDAR apparatus can contain greater than 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45 or 50 solenoids. In some aspects, atleast one solenoid can be open. For example, an open solenoid can beused to allow flow to pass from one part of the microfluidic device to adifferent part of the microfluidic device. In some aspects, greater than1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 solenoids canbe open. In some aspects, one part can be the sample entry point. Insome aspects, one part can be the waste channel. In some aspects, onepart can be a sorting chamber. In some aspects, at least one solenoidcan be normally open (N.O.) In some aspects, greater than 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 solenoids can be normally open.

In some aspects, solenoids can be closed. For example, a closed solenoidcan be used to prevent flow from passing from one part of themicrofluidic device to a different part of the microfluidic device. Insome aspects, at least one solenoid can be closed. In some aspects,greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50solenoids can be closed. In some aspects, one part can be the sampleentry point. In some aspects, one part can be the waste channel. In someaspects, one part can be a sorting chamber. In some aspects, at leastone solenoid can be normally closed (N.C.) In some aspects, greater than1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 solenoids canbe normally closed.

In some aspects, solenoids can be switched from open to closed. Forexample, closing an open solenoid can be used to prevent flow frompassing from one part of the microfluidic device to a different part ofthe microfluidic device. In some aspects, at least one solenoid can beswitched from open to closed. In some aspects, greater than 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 solenoids can be switchedfrom open to closed. In some aspects, one part can be the sample entrypoint. In some aspects, one part can be the waste channel. In someaspects, one part can be a sorting chamber.

In some aspects, solenoids can be switched from closed to open. Forexample, opening a closed solenoid can be used to allow fluid to passfrom one part of the microfluidic device to a different part of themicrofluidic device. In some aspects, at least one solenoid can beswitched from closed to open. In some aspects, greater than 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 solenoids can be switchedfrom closed to open. In some aspects, one part can be the sample entrypoint. In some aspects, one part can be the waste channel. In someaspects, one part can be a sorting chamber.

In some aspects, the structure of the fluidic microfluidic chip and thecorresponding scheme of the hydrodynamic sorting that can be used foreDAR are shown in FIG. 6. The position of the solenoid can be markedwhen there are outlets or inlets on a single channel. For example inFIG. 6F, the position of the second solenoid can be “center waste,”meaning that it is on the center outlet of the waste collection channel.In every scheme, except that shown in FIG. 6G, when the “positive”events can be detected, the DC voltage applied on the solenoids can bechanged to trigger the sorting, and after a certain period of time canbe returned to the normal state. In some aspects, 4 individual steps canbe used to control the sorting (e.g., scheme shown in FIG. 6G). Bothsolenoids can be closed and the fluid can flow to the waste channel. Thesorting can be triggered which could open the solenoid on the collectionside to perform the switch-over step. The other solenoid can be openedto perform the switchback step after the cell is collected. Bothsolenoids can be closed at the same time after the fluid flow wascompletely switched back to revert to the normal state. A summary of thefluidic configuration and performance of eight different hydrodynamicsorting schemes described herein is depicted in Table 1 (below). In someaspects, two solenoids can be used (e.g., schemes shown in FIGS. 6C, 6E,and 6G).

TABLE 1 Summary of the fluidic configuration and performance of 8sorting schemes. Switch Switch Left Right over back Normal pressurepressure time time Scheme Position state (psi) (psi) (ms) (ms) aCollection Closed Low High ~2-3 ~15-25 b Waste Open High Low ~15-20 ~2-3c Collection Closed Low High ~4-5 ~10 Waste Open d Right Buffer CloseLow High  ~3 ~40 e Waste Open High Low ~25  ~2 f Collection Closed LowHigh ~25 ~5-6 Center Waste Open g Collection Closed Low High ~2-3 ~2-3Center Waste Closed h Center Right Closed Low High ~2-3 ~2-3 Buffer

In some aspects, an off-chip solenoid can be used. The off-chip solenoidcan be closed for most of the method. To open the off-chip solenoid,voltage (e.g., 5V DC voltage) can be applied. The off-chip solenoid canopen rapidly (e.g., less than or equal to three milliseconds). Where anoff-chip solenoid is used, the preparation of the microfluidic chip canbe modified and solenoids can be connected with microchannels. In someaspects, an on-chip solenoid can be used. The on-chip solenoid can beclosed for most of the method. To open the on-chip solenoid, voltage(e.g., 5V DC voltage) can be applied. The on-chip solenoid can openrapidly (e.g., less than or equal to three milliseconds). Where anon-chip solenoid is used, the preparation of the microfluidic chip canbe modified and solenoids can be connected with microchannels.

For example, in this scheme, an off-chip solenoid is used. The labeledfluid sample can be injected into the top channel of the microfluidicchip using a syringe pump (FIG. 7a ). Two side channels, where bufferflows through, can be used to control the active sorting step. Two portsare located on the right-side channel, and both are connected to apressurized buffer source. The off-chip solenoid can be connected to theport near the sorting junction to control the hydrodynamic switch. Theswitching process using an off-chip solenoid can be stable and canmaintain stability through 10⁵ on-off cycles.

In some aspects, an in-line solenoid can be placed on the buffer line toprevent the fluid sample from contacting the solenoid. This caneliminate the possibility of sample decay and cross-contamination. Therecan be a constant flow of buffer in the rare cell collection channelduring use of the eDAR apparatus and method. The on-chip solenoid canimprove the efficiency of the subsequent purification (e.g.,purification chamber) step and can prevent the formation of aggregatesof cells.

In some aspects, the flow of fluid in the eDAR apparatus can beregulated with one of the following either upstream or downstream of thedetection volume: a solenoid, a valve, a bubble, an electric field, amagnetic field, an optical field, a pneumatic pressure source, a solidparticle, a membrane, an immiscible droplet, a gravitationaldifferential, or a coating to alter surface tension of the channel. Theflow can be stopped, decelerated, or accelerated as the cells traversethrough the detection volume.

In some aspects, continuous flow of the fluid sample through a flowchannel can be maintained during detection. In some aspects, theindividual aliquots may not be physically separated, but rather can bedefined by an optical detection step and/or a sorting step.

For example, the flow can be directed into the channel that can be usedto collect the waste (FIG. 7b ). There can be two channels after thesorting junction. The left channel can collect positive aliquots,deliver them to the filtration and collection area for furtherpurification (e.g., purification chamber); the right channel collectswaste (e.g., the negative aliquots). In this case, no voltage is appliedthe solenoid when aliquots are ranked as “negative” and remains closed(FIG. 7b ). The change in flow pattern can occur after an initialpressure drop between the No. 1 and 3 buffer sources (FIG. 7a ). Apositive event can be detected by the first detection window. In thiscase, a DC voltage (e.g., 5V) can be applied to the solenoid to open thebuffer flow from the buffer reservoir (e.g., No. 2). The decreased flowresistance of the buffer channel on the right side can generate a higherflow rate. The fluid flow can be pushed from the right side to the leftto collect the positive aliquot (FIG. 7c ). The aliquot can be collectedand confirmed by the second detection window. In some aspects, thesolenoid can be closed to switch the fluid flow back to the wastecollection channel (FIG. 7d ). The time required for the switch-over andback can be less than 20 milliseconds, in an exemplary case the time isor between 2-3 milliseconds (Table 1 and FIG. 8; frame rate=1,918 framesper second). In some aspects, the conditions used can be repeated formore than 10⁵ on-off cycles.

In some aspects, the flow can be delivered by, for example, methods anddevices that induce hydrodynamic fluidic pressure, which includes but isnot limited to those that operate on the basis of mechanical principles(e.g., external syringe pumps, pneumatic membrane pumps, vibratingmembrane pumps, vacuum devices, centrifugal forces, and capillaryaction); electrical or magnetic principles (e.g., electroosmotic flow,electrokinetic pumps piezoelectric/ultrasonic pumps, ferrofluidic plugs,electrohydrodynamic pumps, and magnetohydrodynamic pumps); thermodynamicprinciples (e.g., gas bubble generation/phase-change-induced volumeexpansion); surface-wetting principles (e.g., electrowetting,chemically, thermally, and radioactively induced surface-tensiongradient).

In yet other cases, the fluid can be delivered or channeled by a fluiddrive force provided by gravity feed, surface tension (like capillaryaction), electrostatic forces (electrokinetic flow), centrifugal flow(substrate disposed on a compact disc and rotated), magnetic forces(oscillating ions causes flow), magnetohydrodynamic forces and a vacuumor pressure differential.

In some aspects, fluid flow control devices, such as those enumeratedwith regard to methods and devices for inducing hydrodynamic fluidpressure or fluid drive force, can be coupled to an input port or anoutput port of the present subject matter. In some aspects, multipleports are provided at either or both of the inlet and outlet and one ormore ports are coupled to a fluid flow control device.

Transit Time

The analyte (e.g., rare cell or circulating tumor cell (CTC)) can flowfrom the first detection window to the second detection window. The timethat can elapse between recording of the decision APD peak in the firstwindow and detection of the confirmation signal can be the transit timefor sorting an analyte. In some aspects, the transit time can vary. Insome aspects, the liner flow rate can affect the transit time. In someaspects, the laminar flow of the microchannel can affect the transittime. In some aspects, the volumetric flow rate can be set to 40 μL/min(FIG. 9b ). In some aspects, the volumetric flow rates can be about 1μL/min, 2 μL/min, 3 μL/min, 4 μL/min, 5 μL/min, 6 μL/min, 7 μL/min, 8μL/min, 9 μL/min, 10 μL/min, 11 μL/min, 12 μL/min, 13 μL/min, 14 μL/min,15 μL/min, 16 μL/min, 17 μL/min, 18 μL/min, 19 μL/min, 20 μL/min, 21μL/min, 22 μL/min, 23 μL/min, 24 μL/min, 25 μL/min, 30 μL/min, 35μL/min, 40 μL/min, 45 μL/min, 50 μL/min, 55 μL/min, 60 μL/min, 65μL/min, 70 μL/min, 75 μL/min, 80 μL/min, 85 μL/min, 90 μL/min, 95μL/min, 100 μL/min, or 200 μL/min.

In some aspects, the flow rate can be within the range of about 1μL/min-5 μL/min, 3 μL/min-10 μL/min, 5 μL/min-15 μL/min, 10 μL/min-20μL/min, 15 μL/min-30 μL/min, 20 μL/min-40 μL/min, 30 μL/min-50 μL/min,40 μL/min-60 μL/min, 50 μL/min-70 μL/min, 60 μL/min-80 μL/min, 70μL/min-90 μL/min, 80 μL/min-100 μL/min, 90 μL/min-100 μL/min or 90μL/min-200 μL/min. In other cases, the volumetric flow rate can be setto 80 μL/min (FIG. 9b ).

In some aspects, the volumetric flow rates can be 1 μL/min, 2 μL/min, 3μL/min, 4 μL/min, 5 μL/min, 6 μL/min, 7 μL/min, 8 μL/min, 9 μL/min, 10μL/min, 11 μL/min, 12 μL/min, 13 μL/min, 14 μL/min, 15 μL/min, 16μL/min, 17 μL/min, 18 μL/min, 19 μL/min, 20 μL/min, 21 μL/min, 22μL/min, 23 μL/min, 24 μL/min, 25 μL/min, 30 μL/min, 35 μL/min, 40μL/min, 45 μL/min, 50 μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70μL/min, 75 μL/min, 80 μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100μL/min, or 200 μL/min.

In some aspects, the flow rate can be within the range of 1 μL/min-5μL/min, 3 μL/min-10 μL/min, 5 μL/min-15 μL/min, 10 μL/min-20 μL/min, 15μL/min-30 μL/min, 20 μL/min-40 μL/min, 30 μL/min-50 μL/min, 40 μL/min-60μL/min, 50 μL/min-70 μL/min, 60 μL/min-80 μL/min, 70 μL/min-90 μL/min,80 μL/min-100 μL/min, 90 μL/min-100 μL/min or 90 μL/min-200 μL/min. Ahigher flow rate can be used to achieve a lower transit time (FIG. 9c ).

Detection

The disclosure provides an apparatus for eDAR using a microfluidic chipthat can be equipped with a detection system. In some aspects, thedetection system can include a line-confocal detection scheme. In theline-confocal detection scheme, two laser sources (e.g., 488 and 633 nm)form detection windows (e.g., two) using a series of dichroic mirrors,cylindrical lens and beam splitters. The first detection window can havetwo laser beams simultaneously overlapping and can be used detect thefluorescence signals from the labeled rare cells (e.g., CTCs). Thesecond detection window can be used to confirm the identity of the rarecells, or lack of rare cells, in the sorted aliquots. The seconddetection window can further be used to monitor the sorting efficiency.

In some aspects, two rare particles can be detected simultaneously. Eachrare particle can be contacted with a unique detection reagent and eachdetection reagent can be detected by one of two detection devices.Furthermore, wherein the detection reagents comprise fluorescentmoieties, two interrogation devices (e.g., two lasers) producingradiation at different wavelengths corresponding to excitationwavelengths of the different fluorescent moieties can be used. Therespective fluorescent radiation can be detected by two differentdetection devices. In some aspects, the detection reagents can bedifferentiable by fluorescence at different wavelengths.

In some aspects, the two or more rare particles can be detected inseries. For example, the method can comprise detecting a first rareparticle at a first location of an eDAR apparatus and detecting a secondrare cell at a second location of an eDAR apparatus. In this case, thealiquot in which the first and second particle reside can be channeledto a new location after the first detection step, after the seconddetection step, or after both detection steps.

In certain aspects, the detection event can occur at a regularfrequency. The frequency may relate to the size of the detection volumeand the flow rate of the fluid sample. The detection volume of aparticular apparatus can be within the range of, and including thelimits of, 0.1-100 μL (e.g., 10 μL) and the fluid sample can flowthrough the apparatus at a rate within the range of, and including thelimits of, 1-1000 μL/second (e.g., 100 μL/second), a different aliquotcan be detected within the range of, and including the limits of, onceevery 0.001-1 second (e.g., 0.1 seconds), or at a rate within the rangeof, and including the limits of, 1-100 Hz (e.g., 10 Hz).

In some aspects, the geometry of the apparatus and the volume of thefluid to be processed can affect the rate. For example, aliquots cantraverse through the detection volume at a rate within the range of, andincluding the limits of, 0.1 kHz and 100 MHz. In some aspects, thealiquots traverse through the detection volume at a rate within therange of, and including the limits of, 10 Hz and 10 MHz or about 10 MHz.In some aspects, the aliquots may traverse through the detection volumeat a frequency within the range of, and including the limits of, 0.1 kHzand 100 MHz or about 100 MHz, or within the range of, and including thelimits of 1 kHz or about 1 kHz and 10 MHz or about 10 MHz, or frequencywithin the range of, and including the limits of about 1 kHz and 5 MHzor about 5 MHz, or frequency within the range of, and including thelimits of 1 kHz or about 1 kHz and 1 MHz or about 1 MHz. In someaspects, the frequency by which the aliquots traverse through thedetection volume can be at least about 0.1 kHz, or at least about 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250,300, 400, 500, 600, 700, 800, or 900 kHz, or at least about 1 MHz, or atleast about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,60, 70, 80, 90, or 100 MHz. In some aspects, the frequency by which thealiquots traverse through the detection volume can be at least 0.1 kHz,or at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150,200, 250, 300, 400, 500, 600, 700, 800, or 900 kHz, or at least 1 MHz,or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,60, 70, 80, 90, or 100 MHz.

In some aspects, detection of a characteristic from an ensemble ofanalytes (e.g., cells) in an aliquot of a fluid sample can besimultaneous or cumulative over time. For example, detection of acharacteristic can be simultaneous from a large aliquot containing anensemble of analytes. During simultaneous mode, the particles can becarried by a flow of variable velocity. In other cases, particles can becarried by a steady flow as they traverse through the detection volume.

In various aspects, the eDAR apparatuses and methods allow a signal tobe detected simultaneously from a plurality of analytes (e.g., aplurality of cells). In some aspects, an aliquot is ranked based on asignal detected simultaneously from a plurality of analytes presentwithin the aliquot.

In other cases, the methods herein provide for detection of acharacteristic from an ensemble of cells can emanate over time(“cumulative”) from a small detection volume which is on the order of asingle cell, but with multiple cells traversing through the detectionvolume with the aid of flow. Cumulative mode of eDAR is distinct fromtime-lapse overlay of consecutive signals or frames emanating from asingle bioparticle; timelapse overlay of a single bioparticle does notconstitute an ensemble of bioparticles. In both simultaneous andcumulative, a decision is rendered only after a characteristic from anensemble of cells has been detected.

In some aspects, the detector is selected from the group consisting of acamera, an electron multiplier, a charge-coupled device (CCD) imagesensor, a photomultiplier tube (PMT), an avalanche photodiode (APD), asingle-photon avalanche diode (SPAD), a silicon photomultiplier (SiPM),and a complementary metal oxide semiconductor (CMOS) image sensor. Insome aspects, the eDAR apparatus provided herein can comprise a photo,electro, acoustical or magnetic detector to track the motion of selectcells or to enumerate select particles or cells present in an aliquot.

In some aspects, an apparatus or method provided herein can incorporatefluorescence (single or multi-color) microscopy imaging in variousconfigurations, which include but are not limited to bright-field, epi,confocal, trans, DIC (differential interference contrast), dark-field,Hoffman, or phase-contrast.

In some aspects, the apparatuses provided herein can comprise aplurality of detection devices, for example, at least 2, 3, 4, 5, 6, 7,8, 9, 10, or more detection devices. Multiple detection devices may beuseful for performing the methods of the present disclosure, forexample, wherein more than one rare particle or cell is present in afluid sample, more than one cell marker is being used to differentiatedifferent cell types, or multiple detection reagents are being detectedsimultaneously. In some aspects, the detection devices can include aconfirmatory laser. For example, the confirmatory laser can be used tointerrogate the sorted aliquot. In some aspects, the sorted aliquotdetected by the confirmatory laser can be a positive aliquot andretained after sorting. In some aspects, the sorted aliquot detected bythe confirmatory laser can be a negative aliquot and can be discardedafter sorting. For example, the confirmatory laser can be used as asecond detection method to control the accuracy of the eDAR sortingscheme.

Sources of Radiation and Devices

The disclosure provides an apparatus for eDAR that can include adetection device or an imaging device and a ranking device (e.g., acomputer). In some aspects, a laser (or more than one laser) can serveas an interrogation device. An inverted microscope with photodiodes,photomultipliers, or cameras can be used as a detection device. A maskcan be placed in a path between the channel and the detection devices.

In some aspects, the interrogation device (e.g., a 488 nm solid-statediode pumped laser and a 633 nm HeNe laser) can be directed into aninverted microscope. The two laser beams can be shaped using cylindricaloptics or diffractive optics to form a collimated elliptical beam withan aspect ratio of 10 to 1 prior to entering the microscope objective.Using a combination of half-waveplate and polarizing beam splitter, theintensity of each beam can be adjusted, while mirrors independentlysteer the light to create a spatially co-localized excitation region.The fluorescence from bioparticles can be split into three wavelengthbands by two dichroic mirrors before passing through the bandpassfilters and refocused onto the three single-photon avalanche diodes(SPADs). One SPAD can collect fluorescence in the wavelength range of560-610 nm, a second SPAD cab collect fluorescence in the range of645-700 nm, and a third SPAD can collect in the range of 500-540 nm. TheSPAD outputs are directed to a digital processor (e.g., computer) with acounter/timer board and analyzed with several algorithms.

In some aspects, a digital processor accepts a signal from the detectiondevice and through an algorithm to rank the aliquot. The digitalprocessor can direct the aliquot into the appropriate channel based onthe value of the ranking (e.g., the presence, absence, quantity,identity, or composition of rare particles in the fluid sample). eDARcan consist of one, two, three, four, five or six detection devices andone, two, three, four, five or six interrogation devices, or multitudesof detection devices and interrogation devices.

Dual Capture eDAR

This disclosure further provides an apparatus for a “dual-capture”version of eDAR. The dual-capture apparatus can separate two differentsubpopulations of rare cells from the same sample of mixed fluid. Thiscan be performed on a single microfluidic chip simultaneously. The twosubpopulations can further be trapped separately on two differentregions on the microfluidic chip.

A general structure of the dual capture version of the microfluidic eDARdevice is depicted in FIG. 11. Samples can be labeled with at least twounique antibodies that are conjugated to unique fluorescent tags. Forexample, the sample (e.g., blood) can be labeled with a first tagconjugated with a unique fluorophore that binds to a marker (e.g.,epithelial, anti-EpCAM-PE) on an analyte within the sample and a secondtag conjugated with a unique fluorophore that binds to a marker (e.g.,mesenchymal, anti-EGFR or anti-vimentin) on an analyte within the samplewhere each fluorophore can have a different wavelength of emission(e.g., FITC). The labeled blood sample can be injected into themicrofluidic chip. The rare cells which express EpCAM can be detectedusing the line-confocal scheme (described before) and the peak can bedetected in the yellow channel. A sorting event can be triggered tocollect that particular aliquot into a collection channel (e.g.,collection channel #1). In some aspects, the aliquot can be ranked aspositive to a mesenchymal markers then the rare cell can be sorted in toa different collection channel (e.g., collection channel #2). The twosubpopulations of rare cells can be separately trapped and enriched onthe dual-capture eDAR microfluidic chip.

The dual-capture eDAR apparatus can use a fluidic switching scheme (FIG.11). The labeled sample can be introduced into the main channel on thetop. Buffer can flow in the two side channels to control thehydrodynamic switching of the blood flow using two solenoids. In someaspects, the filtration area of the dual-capture eDAR can be built usingmicroslits (described before). Two subpopulations of CTCs can beseparated and trapped on two different filtration areas on the samemicrofluidic chip.

The dual-capture eDAR apparatus can include solenoids. In some aspects,both of the solenoids can be closed when aliquots are ranked as negative(FIG. 12). The sample can flow to the bottom center channel to collectas waste if the pressure is balanced in both channels. In other cases,when the aliquots are ranked positive with respect to only one of thetwo markers (e.g., epithelial) then solenoid #2 can be opened for thesample to flow into the collection channel on the left (FIG. 12B). Afterthe aliquot is collected, solenoid #2 can be closed again and the flowof the sample is returned to the center channel. In some aspects, thealiquots can be ranked as positive to only one marker (e.g., epithelial)and solenoid #1 can be opened to allow flow of the sample to the rightchannel (FIG. 12C). In some aspects, the response time for the two typesof sorting events in the dual-capture eDAR apparatus is quick (e.g.,less than or equal to 3 milliseconds).

Immunostaining and Bleaching in a Microfluidic Chip

This disclosure provides a scheme for a sequential immunostaining andbleaching method using a microfluidic device. The method can use theeDAR apparatus cited in PCT WO 2010/120818, the apparatus providedherein, or other apparatuses known in the art. In some aspects, thisscheme can be coupled with eDAR. In some aspects, the scheme can becoupled with an in-line staining and washing system to minimize the deadvolume; decrease the amount of antibodies used; avoid introducing airbubbles; and automate the process of isolating, staining and bleachingrare cells.

eDAR can be used to capture analytes (e.g., rare cells). The rare cellscan be enriched to a small area (e.g., chamber) on the microfluidicchip. The small area can be used to rapidly image the labeled cell andminimize the amount of reagents (e.g., antibodies, buffers, etc) used.The microfluidic chip can be designed with an open, accessiblestructure. The open structure can allow for further manipulation ofsingle cells (e.g., picking up a cell of interest or delivering certainreagents to a cell).

In some aspects, a virtual aliquot can be acquired by a combination ofthe laser detection beam, the volumetric flow rate, and the sortingspeed. Based on these factors, the labeled sample can be virtuallydivided up into aliquots (e.g., 500,000 per 1 mL of sample at 2 nL peraliquot). In some aspects, the line-confocal detection method can detectfluorescence emission from each cell. The virtual aliquots can be rankedaccording to the labeling scheme. If labeled, the aliquot is “positive”and if unlabeled, the aliquot is “negative.” In some aspects, negativealiquots can be discarded (FIG. 4A).

In some aspects, following the aliquot ranking, an automatic feedbackmechanism can trigger a hydrodynamic switch of the flow. The switch cancollect and transfer the “positive” aliquots to an area of themicrofluidic chip for further purification (e.g., purification chamber)and analysis. In some aspects, the sorted aliquots can be transferred toan area of the microfluidic chip where rare cells (e.g., circulatingtumor cells) can be trapped and the blood cells can be discarded (FIG.4A). In some aspects, the trapped cells can be imaged and labeled withone or several tags (e.g., labeled antibodies) that recognizebiomarkers.

The labeling and imaging of analytes (e.g., rare cells) can occur on themicrofluidic chip (e.g., eDAR apparatus). In some aspects, two ports onthe microfluidic chip can be placed in the open position to perform theperfusion labeling and washing steps (FIG. 13A). The remaining threeports can remain closed. A peristaltic pump can deliver washing buffer(e.g., Isoton (Beckman Coulter Inc., Chino, CA)) and labeling reagentsto the microfluidic chip. A pressurized buffer source can be coupled tothe pump and microfluidic chip using a six-way valve. In some aspects,the other three ports on the valve can be closed to prevent leak and/orcontamination.

This method provides for staining of markers on isolated analytes (e.g.,rare cells) (FIG. 13B). The method can be performed using antibodies(e.g., less than or equal to 10 nanograms) and an incubation step (e.g.,less than or equal to 30 minutes). To perform the method ofimmunostaining and photobleaching, the six-way valve can be turned opentowards the pressurized buffer for stable control of the hydrodynamicswitching. The valve can be turned towards the peristaltic-pump toinject specific amounts of a reagent in to the microfluidic chip. Aperistaltic pump can be used to deliver the labeling reagents andwashing buffer (FIG. 13). The cross bars indicate the correspondingports can be closed. In some aspects, the valve can prevent air bubblesfrom entering the microfluidic chip system.

In some aspects, the method can be used to perform intracellular markerstaining. In this method, the captured cells can be fixed andpermeabilized using a permeabilization agent, such as a detergent (e.g.,Triton, surfynol 465 surfactant, etc.) on the microfluidic chip prior toimmunostaining. The method further describes multiple rounds ofimmunostaining the individual cells, washing of the stained cells,imaging of the markers in the cells and photobleaching of the markersthat are bound to the individual cells. The steps can be repeatedsequentially for multiple rounds.

In some aspects, isolated cells can be fixed. Fixation can be performedusing perfusion (e.g., manual or automatic) or diffusion (e.g., manualor automatic) methods. In some aspects, fixation can be performed on themicrofluidic chip or off the microfluidic chip after the sample has beenremoved from the microfluidic chip. In some aspects, samples or isolatedparticles may not be fixed. In this case, samples may include livingparticles (e.g., mammalian cells, bacterial cells, fungal cells, yeastcells, etc.). Samples may include a structure that may be damaged byfixation.

In some aspects, isolated particles can be permeabilized.Permeabilization can be performed using perfusion (e.g., manual orautomatic) or diffusion (e.g., manual or automatic). In some aspects,permeabilization can be performed on the microfluidic chip or off themicrofluidic chip after the sample has been removed from themicrofluidic chip. In some aspects, samples or isolated particles maynot be permeabilized. In this case, samples may include markers (e.g.,extracellular, cell-associated, etc.,). Samples may include a structurethat may be damaged by permeabilization.

Analytes that have been contacted with a tag can be photobleached. Theprocess of photobleaching can reduce the signal emitted from the tagthat is in contact with the analyte. In some aspects, a device that canbe used for photobleaching can include, a laser, a light emitting diode,a voltaic arc lamp, an incandescent lamp, a fluorescent lamp, anultraviolet lamp or a halogen lamp. In some aspects, the voltaic arclamp can be selected from, but is not limited to, the following types ofarc lamps, neon, argon, xenon, krypton, sodium, metal halide, mercury orcarbon.

In some aspects, analytes can be contacted with a tag and the tag can bebleached chemically. In some aspects, the tag can generate a signal thatis readable by a detector. For example, the signal can be emitted byfluorophores. In some aspects, the signal can be chemically quenched orbleached using chemicals including, but not limited to, oxidizingreagents, halogen ions, reducing agents, or any other appropriatefluorescence quencher. In some aspects, the reducing agent isdithiothreitol. In other cases, the tag that can recognize thebiomarkers, such as the antibodies, can be chemically dissociated fromthe binding sites with a high efficiency, using appropriate reagents. Insome aspects, the destaining reagent can be a Tris-based buffer with 2%SDS, 20 mM di-thiothreitol (DTT) and 60 mM Tris pH 6.8.

The method can include photobleaching after each imaging step. Theparameters used can deliver a highly efficient and rapid throughputphotobleaching process (FIG. 14 and Table 2). Photobleaching curves forcells (e.g., MCF-7) labeled with a tag directed to a biomarker (e.g.,anti-EpCAM-PE) can be generated after exposure of labeled cells todifferent powers of the photobleaching light source (FIG. 15A).Additional curves can be generated using multiple conjugates (e.g.,Alexa 647, Alexa488, FITC, PE) to the same antibody (FIG. 15B).

The steps of labeling and photobleaching can be repeated multiple timesto study several groups of biomarkers. For example, two biomarkers ofinterest can be combined with a positive control marker (e.g., nuclearstain) and a negative control marker (e.g., CD45) to create a group. Onegroup can be studied in each round, followed by photobleaching before asecond round of immunostaining occurs. In some aspects, the method ofsequential immunostaining and photobleaching can be used to determinethe expression of different protein markers on an analyte. For example,an analyte (e.g., rare cell, cancer cell, etc.) trapped on themicrofluidic chip is EpCAM, cytokeratin and Hoescht positive but CD45negative (Table 2, below).

TABLE 2 Shows experimental details of four rounds of immunostaining andphotobleaching. Yellow Anti-EpCAM-PE MUC1-PE Anti-CD24 PE Anti-CD166 PEchannel Lot# 515776 Lot# B160021 Lot# B159732 Lot# B139297 (1:50dilution, (1:50 dilution, (1:50 dilution, (1:10 dilution, Biolegend, SanBiolegend, San Biolegend, San Biolegend, San Diego, CA) Diego, CA)Diego, CA Diego, CA) Red (PAN) Cytokeratin- HER2- Anti-CD44- EGFR-APCChannel AlexaFluro647 AlexaFluro647 AlexaFluro647 Lot# B161059 Lot#4528S-14 Lot# B110523 Lot# B124953 (1:40 dilution, (1:10 dilution, (1:50dilution, (1:66 dilution, Biolegend, San CellSignalling, Biolegend, SanBiolegend, San Diego, CA) Danvers, MA) Diego, CA) Diego, CA) BlueHoechst Hoechst Hoechst Hoechst Channel Lot# 1249542 Lot# 1249542 Lot#1249542 Lot# 1249542 (1:500 dilution, Life (1:500 dilution, Life (1:500dilution, Life (1:500 dilution, technologies, technologies,technologies, Life technologies, Carlsbad, CA) Carlsbad, CA) Carlsbad,CA) Carlsbad, CA) Green Anti-CD45-FITC Anti-CD45-FITC Anti-CD45-FITCAnti-CD45-FITC Channel Lot# B116314 Lot# B116314 Lot# B116314 Lot#B116314 (1:66 dilution, (1:66 dilution, (1:66 dilution, (1:66 dilution,Biolegend, San Biolegend, San Biolegend, San Biolegend, San Diego, CA)Diego, CA) Diego, CA) Diego, CA)

In some aspects, only one first tag that is directed to a biomarker canbe used in the first round of immunostaining. In this case, the firsttag can be photobleached. A different second tag that is directed to adifferent biomarker can be used in a next round of immunostaining. Thedifferent second tag can be photobleached.

In some aspects, only one first tag that is directed to a biomarker canbe used in the first round of immunostaining. In this case, the firsttag can be photobleached. The same second tag that is directed to adifferent biomarker can be used in a next round of immunostaining. Thesecond same tag can be photobleached.

In some aspects, only one first tag that is directed to a biomarker canbe used in the first round of immunostaining. In this case, the firsttag can be photobleached. A different second tag that is directed to adifferent biomarker can be used in a next round of immunostaining. Thesecond different tag can be photobleached.

In some aspects, only one first tag that is directed to a biomarker canbe used in the first round of immunostaining. In this case, the firsttag can be photobleached. A different second tag that is directed to asame biomarker can be used in a next round of immunostaining. The seconddifferent tag can be photobleached.

In some aspects, a plurality of tags comprise a first set where each aredirected to a unique biomarker can be used in the first round ofimmunostaining. In this case, the first set of a plurality of tags arephotobleached. A different second set of a plurality of tags that aredirected to a different set of unique biomarkers can be used in a nextround of immunostaining. The second set of different tags can bephotobleached. In some aspects, some of the tags used in the first setcan also be used in the second set. In some aspects, some of thebiomarkers targeted by the tags in the first set can also be targeted bythe tags in the second set. In some aspects, the second set ofbiomarkers can be identical to the first set of biomarkers. In someaspects, the second set of tags can be identical to the first set oftags. In some aspects, the second set of tags can contain overlappingtags with the first set of tags. In some aspects, the second set ofbiomarkers can contain overlapping biomarkers to the first set ofbiomarkers.

In another case, six (e.g., multiple) individual cells (e.g., cancercells) can be trapped on a microfluidic chip (FIG. 14). The eightbiomarkers can include two control biomarkers (e.g., one positivecontrol and one negative control) and four biomarkers (e.g.,EpCAM/Cytokeratin, MUC1/Her2, CD44/CD24 and CD166/EGFR) can be observedper round. In some aspects, the positive control biomarker (e.g.,Hoechst nuclear stain) may not be photobleached. The negative controlbiomarker (e.g., CD45) can be photobleached and a new tag can beincluded with each round of immunostaining. The four biomarkers perround can include both controls and two additional markers. Each roundcan include immunostaining, imaging and photobleaching (Table 2).

An in-line immunostaining and photobleaching system can be coupled withthe method of immunostaining and photobleaching for the labeling andfluorescence imaging of analytes (e.g., rare cells). The in-line systemcan increase the speed and efficiency of the immunostaining andphotobleaching method when multiple rounds are used. The system caninclude, labeling rare cells (e.g., CTCs) with a group of antibodiesconjugated to different fluorophores followed by photobleaching. Afterphotobleaching, the rare cells can be re-labeled with differentfluorescent antibodies against additional biomarkers.

In some aspects, rare cells isolated using the eDAR microfluidic chipapparatus can be washed using buffers. The buffers can be any buffersuitable for the application. Isolated cells can remain on themicrofluidic chip and the main, side and waste channels can be closed byturning off the in-line valves.

The method provides for detection of a signal emitted by a tag, orsignals emitted by several tags, using a device. In some aspects, thedevice can be microscope. The microscope (e.g., confocal, inverted etc.)can be equipped with light sources and detectors for fluorescence. Inother cases, the device can be one of several devices known to those ofskill in the art for detection of signals emitted by tags.

In some aspects, detection can occur in conjunction with photobleaching,after photobleaching is complete, or before photobleaching begins.Detection can be used to determine the end point of emission ofdetectable signal from the tag. In some aspects, detection can occur inconjunction with immunostaining. Detection can also occur after the washstep, before the wash step, during the wash step or in the absence of awash step. In some aspects, detection can also occur after thepermeabilization step, before the permeabilization step, during thepermeabilization step or in the absence of a permeabilization step. Insome aspects, detection can also occur after the fixation step, beforethe fixation step, during the fixation step or in the absence of afixation step.

The method includes imaging of signals emitted by the tags. Devices usedfor imaging have been described above, are included in PCTWO20120/120818 or are known to those of skill in the art. For example,fluorescent images can be collected before and after the photobleachingstep. Images can include data from all emission channels. In someaspects, the emission channels can include, yellow (555 to 605 nm), blue(435 to 485 nm), green (510 to 540 nm) and red (665 to 695 nm). In someaspects, isolated analytes can be imaged using bright-field or Nomarskimicroscopy.

In some aspects, images can be analyzed for expression of multiplebiomarkers. Individual cells (e.g., four) captured by eDAR and contactedwith tags to detect biomarkers (e.g., Her2 and MUC1) and a positivecontrol biomarker (e.g., Hoechst) can be imaged using fluorescence (FIG.16). A digital processor (e.g., computer) and software can be used tomerge multiple images into a single multi-color image (FIG. 16D).

The method further provides for the collection of analytes (e.g.,particles). The particles (e.g., rare cells) collected may have beensubject to immunostaining and photobleaching. In some aspects, thetrapped rare cells can be subject to additional rounds of immunostainingand photobleaching off the microfluidic chip. In some aspects, the rarecells can be collected for further processing. Collection can involveplacement of the rare cells into a reservoir (e.g., tube, plate, array,etc.). Rare cells can be fixed after collection. In some aspects, therare cells can be living cells and maintained in cell culture.

In some aspects, the methods provided herein can further be coupled toan assay protocol following isolation of the analytes. Non-limitingexamples of assays that can be coupled to the methods provided hereininclude nucleic-acid based methods such as RNA extraction (with orwithout amplification), cDNA synthesis (reverse transcription), genemicroarrays, DNA extraction, Polymerase Chain Reactions (PCR) (single,nested, quantitative real-time, or linker-adapter), or DNA-methylationanalysis; cytometric methods such as fluorescence in situ hybridization(FISH), laser capture microdissection, flow cytometry, fluorescenceactivated cell sorting (FACS), cell culturing, or comparative genomichybridization (CGH) studies; chemical assay methods such aselectrophoresis, Southern blot analysis or enzyme-linked immunosorbentassay (ELISA); assays to determine the microRNA and siRNA contents;assays to determine the DNA/RNA content; assays to determine lipidcontents; assays to determine carbohydrate contents; assays to determinemetabolite contents; assays to determine protein contents; andfunctional cell assays (e.g., apoptotic assays, cell migration assays,cell proliferation assays, cell differentiation assays, etc.), and thelike.

Types of Labels

The method of immunostaining and photobleaching can include the use oftags, or labels, to identify biomarkers located on trapped analytes(e.g., particles). In some aspects, tags can be detected using the eDARapparatus described herein, or other apparatuses configured fordetection of tags known to those of skill in the art. In some aspects,tags can be photobleached using the components of the apparatusdescribed herein or using a photobleaching device known to those ofskill in the art. In some aspects, the tags can be photobleached usingan apparatus that may cause damage to the particle. In other cases, tagscannot be photobleached.

In some aspects, tags can be used as labels for immunostaining. Tags canbe detectable by spectroscopic, photochemical, biochemical,immunochemical, chemical, or other physical means. For example, usefultags can include, without limitation radionuclides, fluorescent dyes(e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™,rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5,etc.), fluorescent markers (e.g., green fluorescent protein (GFP),phycoerythrin (PE), etc.), autoquenched fluorescent compounds that areactivated by tumor-associated proteases, enzymes (e.g., luciferase,horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles,biotin, digoxigenin, and the like. In some aspects, the tags can emit ina spectrum detectable as a color in an image. The colors can includered, blue, yellow, green, purple, orange and the like.

In some aspects, the tags can be perfused to selectively label oraccentuate the isolated cells. Examples of such reagents include,without limitation, fluorescent, immunofluorescent, dye-conjugatedmolecules (such as antibodies, fab fragments, aptamers, polymers,ligands, agonists, antagonists, or combinations thereof) magnetic,electroactive, bioactive, or photoactive compounds. An example is to usea stain that reacts with cytokeratins, which are integral components ofthe cytoskeleton in epithelial cancerous cells. Other dye examplesinclude fluorescein isothiocyanate (FITC)-conjugated mouse anti-humanepithelial antibody (HEA) and phycoerythrin (PE)-conjugated anti-CD45.Other examples of dye-conjugated antibodies include but are not limitedto the pan-cytokeratin antibody A45B/B3, AE1/AE3, or CAM5.2(pan-cytokeratin antibodies that recognize Cytokeratin 8 (CK8),Cytokeratin 18 (CK18), or Cytokeratin 19 (CK19) and ones against: breastcancer antigen NY-BR-1 (also known as B726P, ANKRD30A, Ankyrin repeatdomain 30A); B305D isoform A or C (B305D-A ro B305D-C; also known asantigen B305D); Hermes antigen (also known as Antigen CD44, PGP1);E-cadherin (also known as Uvomorulin, Cadherin-1, CDH1);Carcino-embryonic antigen (CEA; also known as CEACAMS orCarcino-embryonic antigen-related cell adhesion molecule 5); β-Humanchorionic gonadotophin (β-HCG; also known as CGB, Chronic gonadotrophin,β polypeptide); Cathepsin-D (also known as CTSD); Neuropeptide Yreceptor Y3 (also known as NPY3R; Lipopolysaccharide-associatedprotein3, LAP3, Fusion; Chemokine (CXC motif, receptor 4); CXCR4);Oncogene ERBB1 (also known as c-erbB-1, Epidermal growth factorreceptor, EGFR); Her-2 Neu (also known as c-erbB-2 or ERBB2); GABAreceptor A, pi (π) polypeptide (also known as GABARAP, GABA-A receptor,pi (π) polypeptide (GABA A(π), γ-Aminobutyric acid type A receptor pi(π) subunit), or GABRP); ppGalNac-T(6) (also known asβ-1-4-N-acetyl-galactosaminyl-transferase 6, GalNActransferase 6,GalNAcT6, UDP-N-acetyl-d-galactosamine:polypeptideN-acetylgalactosaminyltransferase 6, or GALNT6); CK7 (also known asCytokeratin 7, Sarcolectin, SCL, Keratin 7, or KRT7); CK8 (also known asCytokeratin 8, Keratin 8, or KRT8); CK18 (also known as Cytokeratin 18,Keratin 18, or KRT18); CK19 (also known as Cytokeratin 19, Keratin 19,or KRT19); CK20 (also known as Cytokeratin 20, Keratin 20, or KRT20);Mage (also known as Melanoma antigen family A subtytpes or MAGE-Asubtypes); Mage3 (also known as Melanoma antigen family A 3, or MAGA3);Hepatocyte growth factor receptor (also known as HGFR, Renal cellcarninoma papillary 2, RCCP2, Protooncogene met, or MET); Mucin-1 (alsoknown as MUC1, Carcinoma Antigen 15.3, (CA15.3), Carcinoma Antigen 27.29(CA 27.29); CD227 antigen, Episialin, Epithelial Membrane Antigen (EMA),Polymorphic Epithelial Mucin (PEM), Peanut-reactive urinary mucin (PUM),Tumor-associated glycoprotein 12 (TAG12)); Gross Cystic Disease FluidProtein (also known as GCDFP-15, Prolactin-induced protein, PIP);Urokinase receptor (also known as uPR, CD87 antigen, Plasminogenactivator receptor urokinase-type, PLAUR); PTHrP (parathyroldhormone-related proteins; also known as PTHLH); BS106 (also known asB511S, small breast epithelial mucin, or SBEM); Prostatein-likeLipophilin B (LPB, LPHB; also known as Antigen BU101, Secretoglobinfamily 1-D member 2, SCGB1-D2); Mammaglobin 2 (MGB2; also known asMammaglobin B, MGBB, Lacryglobin (LGB) Lipophilin C (LPC, LPHC),Secretoglobin family 2A member 1, or SCGB2A1); Mammaglobin (MGB; alsoknown as Mammaglobin 1, MGB1, Mammaglobin A, MGBA, Secretoglobin family2A member 2, or SCGB2A2); Mammary serine protease inhibitor (Maspin,also known as Serine (or cystein) proteinase inhibitor Glade B(ovalbumin) member 5, or SERPINBS); Prostate epithelium-specific Etstranscription factor (PDEF; also known as Sterile alpha motif pointeddomain-containing ets transcription factor, or SPDEF); Tumor-associatedcalcium signal transducer 1 (also known as Colorectal carcinoma antigenC017-1A, Epithelial Glycoprotein 2 (EGP2), Epithelial glycoprotein 40kDa (EGP40), Epithelial Cell Adhesion Molecule (EpCAM),Epithelial-specific antigen (ESA), Gastrointestinal tumor-associatedantigen 733-2 (GA733-2), KS1/4 antigen, Membrane component of chromosome4 surface marker 1 (M4S1), MK-1 antigen, MIC18 antigen, TROP-1 antigen,or TACSTD1); Telomerase reverse transcriptase (also known as Telomerasecatalytic subunit, or TERT); Trefoil Factor 1 (also known as BreastCancer Estrogen-Inducible Sequence, BCEI, Gastrointestinal TrefoilProtein, GTF, pS2 protein, or TFF1); folate; or Trefoil Factor 3 (alsoknown as Intestinal Trefoil Factor, ITF, p1.B; or TFF3), or the like.

Markers/Biomarkers

The method provides for a plurality of biomarkers that can be expressedby, located on or near an analyte (e.g., trapped particle). Theplurality of biomarkers that can be detected by the instant disclosureis subject to the number of rounds of immunostaining and photobleachingthat can be performed on the trapped particle. Each round ofimmunostaining can include no tags, 1 tag, 2 tags, 3 tags, 4 tags, 5tags, 6 tags, 7 tags, 8 tags, 9 tags or 10 tags. The range of tags foreach round of immunostaining can include 1-10 tags (e.g., 4).

In some aspects, the presence of a marker, such as a biomarker, isindicated by a signal emitted by a tag, wherein the tag has an affinityfor the marker. As used herein, the phrase “has an affinity for” broadlyencompasses both direct molecular binding affinity for a marker by atag, as well as indirect affinity, such as an ability of the tag and themarker to interact via a molecular complex involving one or more otherstructures. For example, in some aspects, the tag may be a primaryantibody with a direct affinity for the marker, whereas in otheraspects, the tag may be a secondary antibody with an indirect affinityfor the marker.

In some aspects, cells are isolated according a specific marker orbiomarker profile, such that a given cell exhibits a unique and/oridentifiable marker or biomarker profile. In certain aspects, the markeror biomarker profile is a specific combination of one or more, two ormore, three or more, four or more, five or more, six or more, seven ormore, eight or more, nine or more, or ten or more markers or biomarkers,which can be used to define and identify a cell or cell type. In someaspects, the marker or biomarker profile can be used to identify a cellas being part of a particular population of cells have a particular setof properties.

In some aspects, biomarkers are extracellular. In other cases,biomarkers can be intracellular, cytoplasmic, intra-cytoplasmic, cellsurface, extranuclear, intranuclear, lysosomal, mitochondrial,endoplasmic reticular and the like. In other cases, biomarkers can beattached to but not in trans to the particle.

In some aspects, biomarkers can be an amino acid, a peptide, apolypeptide, a protein, a denatured protein. In these cases, structurescan be native or denatured.

In some aspects, biomarkers can be a nucleic acid, an oligonucleic acid,a ribonucleic acid, a transfer ribonucleic acid, a messenger ribonucleicacid, a micro ribonucleic acid or a deoxyribonucleic acid. In thesecases, acids can be single or double stranded.

In other cases, the rare particle can be a cell, protein, proteincomplex, nucleic acid, nucleoprotein complex, carbohydrate, metabolite,catabolite, and the like. In some aspects, the rare particle is a cell.In some aspects, the cell can be a cancer cell, a circulating tumor cell(CTC), a cancer stem cell, a cancer cell displaying a cancer surfaceantigen, for example, one selected from the groups consisting of CD44,CD2, CD3, CD10, CD14, CD16, CD24. CD31, CD45, CD64, CD140b, or acombination thereof.

In some aspects the cell is a fetal cell. In some aspects, the fetalcell can be in maternal fluid. The maternal fluid can include wholeblood, fractionated blood, serum, plasma, sweat, tears, ear flow,sputum, lymph, bone marrow suspension, lymph, urine, saliva, semen,vaginal flow, feces, transcervical lavage, cerebrospinal fluid, brainfluid, ascites, breast milk, vitreous humor, aqueous humor, sebum,endolympth, peritoneal fluid, pleural fluid, cerumen, epicardial fluid,and secretions of the respiratory, intestinal and genitourinary tracts.

In some aspects, the analyte is a dissociated cell that is not connectedto other cells or extracellular matrix, or embedded within a tissue.

In some aspects, cancer stem cells can be distinguished from ordinarycancer cells by perfusing other reagents that selectively bind tobiomarkers, which can include but are not limited to CD44, CD2, CD3,CD10, CD14, CD16, CD24. CD31, CD45, CD64, CD140b or CD166.

For example, tags can be designed to detect expression of EGFR and CD166to demonstrate the mesenchymal characteristics of tumor cells. Otherrelated markers, such as vimentin and cadherin, can be used in thisgroup.

In other cases, biomarkers that indicate the viability of the particlecan be used. In these cases, biomarkers which indicate apoptosis,necrosis, mitosis, stage of mitosis, meiosis and the like can be used.

In some aspects, each set of markers can include a nuclear stain(Hoechst) as a positive control biomarker, CD45 conjugated with FITC asa negative control biomarker, and two protein biomarkers conjugated withPE or Alexa 647. In some aspects, the Hoechst stain may not bephotobleached. In this case, Hoechst is a positive control. In thiscase, Hoechst may not be photobleached with a standard light source. Inthis case, a UV exposure may be used to bleach the stain.

Photobleaching

The method provides for photobleaching after immunostaining.Photobleaching can involve use of light to remove or reduce theintensity of the signal emitted by detectable portion of a tag from ananalyte. The tag can be removed by quenching the signal that the tag orlabel emits. In some aspects, the photobleaching can occur after oneround of immunostaining or after sequential rounds of immunostaining.

In some aspects, photobleaching can be performed using a LED or a xenonarc lamp as the light source (Sutter instrument, Novato, Calif.). Usingthe xenon arc lamp, each photobleaching step can occur for 1-59 seconds,1-30 minutes or for 15 minutes.

In some aspects, the maximum power of the LED xenon arc lamp can belocked for safety within a range of 1-20 mW or at 10 mW or at 100 mW or1 W, depending on the power density (e.g., W/m²) of the light source.Use of protective methods, such as wearing protective goggles orcovering the photobleaching area with a black box, can be considered.

In some aspects, the fluorescent emission of a fluorphore (e.g., PE,FITC and Alexa 488) can be bleached. Bleaching the signal (e.g., to lessthan 10%) can occur in a short amount of time (e.g., in less than 5min). Use of lasers with longer wavelengths (e.g., 610 to 660 nm) mayhave a longer duration of time of photobleaching than those of shorterwavelengths (FIG. 17B). In some aspects, when use of multiple lasers atvarious wavelengths is involved, the bleaching time may be higher thanthe minimum requirement for one of the lasers (e.g., 15 min) to achievea high bleaching efficiency with an acceptable throughput.

For example, a curve for photobleaching using different exposure powersusing MCF-7 cells labeled with anti-EpCAM-PE, and placed on a No. 2coverslip is shown in FIG. 15A. Different power settings (e.g., three)can be used to bleach the cells. In some aspects, at a high power (e.g.,greater than 2 mW), the exposure time may be reduced (e.g., less than 10min) to achieve a high (e.g., 95%) bleaching efficiency.

Samples

In the disclosure provided herein, samples can include fluid samples.Fluid samples can originate from a variety of sources. In some aspects,the sources may be humans, mammals, animals, microbes, bacteria, fungus,yeast, viruses, rodents, insects, amphibians and fish.

Fluid samples provided in this disclosure can be liquids that may or maynot contain a rare particle of interest. In some aspects, the sample maybe an environmental fluid sample, for example from a lake, river, ocean,pond, stream, spring, marsh, reservoir, or the like. In yet other cases,the sample may be a water sample, for example from a desalinizationplant, water treatment plant, reservoir, spring, stream, glacial waterflow, water tower, or other water source that may be contemplated as asource of potable water.

In some aspects, this disclosure provides analytes, including analytesthat are rare particles. A rare particle can be a cell or macromoleculepresent in a fluid sample at a low level. In certain aspects, a rareparticle can be a cell, protein, protein complex, nucleic acid,nucleoprotein complex, carbohydrate, metabolite, catabolite, and thelike. In certain aspects, a particle can be considered rare if it ispresent in a fluid sample at a concentration of less than about 10% ofthe total particle population in the fluid, or at less than about 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less of thetotal particle population in the fluid. In yet other cases, the rareparticle can be present in a fluid sample at less than about 1 part per10³ of the total particle population in the fluid, or at less than about1 part per 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², or less ofthe total particle population in the fluid. In certain aspects, aparticle can be considered rare if it is present in a fluid sample at aconcentration of less than 10% of the total particle population in thefluid, or at less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%,0.05%, 0.01%, or less of the total particle population in the fluid. Inyet other cases, the rare particle can be present in a fluid sample atless than 1 part per 10³ of the total particle population in the fluid,or at less than 1 part per 10⁴, 10⁵, 10⁶, 10⁷, 10 ⁸, 10⁹, 10¹⁰, 10¹¹,10¹², or less of the total particle population in the fluid.

In some aspects, a fluid sample can contain a certain percentage ofwater and/or other elements. For example, a fluid sample can be bloodwhich contains, amongst other materials, plasma. Generally, plasma ismostly water and can contain proteins, ions, vitamins, enzymes,hormones, and other chemicals in the body.

In some aspects, the fluid samples can be body fluids. Body fluids areoften complex suspensions of biological particles in a liquid. Forexample, a body fluid can be blood. In some aspects, blood can includeplasma and/or cells (e.g., red blood cells, white blood cells,circulating rare cells) and platelets. In some aspects, 55% of bloodfluid volume can be cells. In some aspects, a blood sample can beconcentrated so that at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, or at least 90% of the blood is cells.In some aspects, a blood sample contains blood that has been depleted ofone or more cell types. In some aspects, a blood sample contains bloodthat has been enriched for one or more cell types. In some aspects, ablood sample can be diluted so that at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, or at least 90% of theblood is cells. In some aspects, a blood sample contains aheterogeneous, homogenous or near-homogenous mix of cells.

Body fluids can include, but are not limited to, whole blood,fractionated blood, serum, plasma, sweat, tears, ear flow, sputum,lymph, bone marrow suspension, lymph, urine, saliva, semen, vaginalflow, feces, transcervical lavage, cerebrospinal fluid, brain fluid,ascites, breast milk, vitreous humor, aqueous humor, sebum, endolympth,peritoneal fluid, pleural fluid, cerumen, epicardial fluid, andsecretions of the respiratory, intestinal and genitourinary tracts. Insome aspects, body fluids can be in contact with various organs (e.g.,lung) that contain mixtures of cells and bioparticles.

In some aspects, body fluids can contain rare particles. In someaspects, the rare particle is a rare cell. Rare cells can be nucleatedor non-nucleated. Rare cells include, but are not limited to, cellsexpressing a malignant phenotype; fetal cells, such as fetal cells inmaternal peripheral blood, tumor cells, such as tumor cells which havebeen shed from tumor into blood or other bodily fluids; cells infectedwith a virus, such as cells infected by HIV, cells transfected with agene of interest; and aberrant subtypes of T-cells or B-cells present inthe peripheral blood of subjects afflicted with autoreactive disorders.

In some aspects, the body fluid can be blood and contain rare cells. Therare cells can be erythrocytes, white blood cells, leukocytes,lymphocytes, B cells, T cells, mast cells, monocytes, macrophages,neutrophils, eosinophils, dendritic cells, stem cells, erythroid cells,cancer cells, tumor cells or cell isolated from any tissue originatingfrom the endoderm, mesoderm, ectoderm or neural crest tissues. Rarecells can be from a primary source or from a secondary source (e.g., acell line).

The type and amount of cells and bioparticles that are present in aparticular body fluid (e.g., blood) can reveal information about thehealth of the organism. For example, a subject suffering from cancer canhave cancerous cells in body fluid. A sample from a subject sufferingfrom an infection can contain an increased number of immune cells (e.g.,lymphocytes, neutrophils, B cells, T cells, dendritic cells, etc.)indicative of infection or can contain pathogenic material such asmicrobial cells or nucleic acids. A sample from a pregnant female cancontain fetal cells indicative of the genotype or karyotype of thefetus.

Some cells or bioparticles can be present in a body fluid in rarequantities compared to normal quantities. Such rare cells can include,circulating tumor cells, cancer stem cells, etc. These cells can appearin a body fluid as a result of an event (e.g., cancer) occurring in aregion of the body. The cell can originate from the organ containing thebody fluid or an organ near the body fluid. For example, the rare breastcancer cell can be present in blood or lymph fluid located near amammary gland. In some aspects, the cell originates from a more distantorgan. For example, a cell that originates from a testicular tumor canbe present in a blood sample taken from the arm.

Analysis

The disclosure further provides for methods whereby an analysis can beperformed. In some aspects, the analysis can include a source forinterrogating an aliquot. In other cases, wherein an aliquot contains ananalyte (e.g., rare particle) that can intrinsically exhibitchemiluminescence or bioluminescence, the apparatus may not require asource for interrogating the aliquot.

In some aspects, a ranking device can be selected from a computer, aprocessor, a controller, a chip with integrated circuits, a circuitboard, an electronic element, software, an algorithm, or a combinationthereof. In some aspects, the processor can be a digital processor(e.g., FGPA, ASIC, or RT). In some aspects, a computer accepts thesignal from the detection device and through an algorithm ranks thealiquot. The ranking device can direct the aliquot into the appropriatechannel based on the value of the ranking (e.g., the presence, absence,quantity, identity, or composition of rare particles in the fluidsample).

In some aspects, the information coming from the first detector can beanalyzed. The information from the first detector can be analyzed andthe result can show that the analyte is positive. In some aspects, theinformation from the detector can be analyzed and the result can showthat the analyte is negative. In some aspects, the ranking device sendsthe signal, which reads to the positive or the negative analyte, to thehydrodynamic switching element (e.g., solenoid valve) to sort theanalytes. In some aspects the positive analyte can be moved to a uniquechannel of the microfluidic chip. In some aspects, the positive analytecan be moved to a unique chamber of the microfluidic chip. In someaspects, the positive analyte can be moved to the waste chamber of themicrofluidic chip. In some aspects the negative analyte can be moved toa unique channel of the microfluidic chip. In some aspects, the negativeanalyte can be moved to a unique chamber of the microfluidic chip. Insome aspects, the negative analyte can be moved to the waste chamber ofthe microfluidic chip. In some aspects, the positive analyte ispartitioned from the negative analyte.

In some aspects, analysis can be performed using the information relayedfrom a second detector. The second detector can be used to confirm theinformation obtained using the first detector. In some aspects, thesecond detector can be located on the microfluidic chip. In someaspects, the second detector may not be located on the microfluidicchip. In some aspects, the second detector obtains a signal from thecells that can be sorted based on the signal detected by the firstdetector. In some aspects, the second detector receives detects signalfrom the sorted cells and can send information about the signal to acomputer. The computer can be used to determine whether the appropriatecell was sorted.

In some aspects, an analysis of the information acquired during themethod of immunostaining can be performed. The method of immunostainingof an analyte occurs in a chamber of the microfluidic chip. At thechamber, a detector can detect one or a plurality of signals from firstround of staining. In some aspects, one or the plurality of signals canbe photobleached. The detector can monitor the duration and intensity ofthe signal emitted. In some aspects, the analyte can undergo a secondround of staining. The detector can detect signal emitted by the analytefollowing the second round of staining. In some aspects, one or theplurality of signals can be photobleached. The detector can monitor theduration and intensity of the signal emitted. In some aspects, thecycles of immunostaining and photobleaching can be repeated a pluralityof cycles. A computer can be used to, for example, compare the one ormore signals of immunostained analytes with first round ofimmunostaining to ensure that photobleaching occurred.

Single-Analyte Array Devices and Methods

This disclosure provides methods and apparatuses for capturing singleanalytes from a liquid phase. In some aspects, the analytes can beparticles. In other cases, the analytes can be cells. The analytes canbe trapped in chambers. In other cases, the analytes can be trapped inwells. In other cases, the analytes can be trapped on patches. In someaspects, the chambers or the wells can be arranged in a linear format.In other cases, the chambers or wells can be arranged in an arrayformat.

The device can be designed in various formats. For example, atwo-dimensional (2D) device can be created. In some aspects, the 2Ddevice can have axially linear flow (FIG. 17B). The axial linear flowcan be a serial-flow design. In some aspects, a three-dimensional (3D)device can be created. For example, the 3D device can have chambersarranged in a grid (FIG. 17C). There are several alternative designsthat can be used for either 2D or 3D strategies if needed.

The 2D device can have channels and chambers with a variety of potentialdimensions. The dimensions can affect the serial-flow resistance trapdesign. A schematic of a device with potential trapping densities anddimensions is shown in FIG. 18 and the magnified region of the device inthe inset. In some aspects, the potential dimensions can include, thewidth of the main channel, the width of the constriction, the lengthfrom the main channel constriction entrance to the main channelconstriction exit, the length across the constriction, as well as theheights of the main channel, the constriction, and the constrictionchamber.

In some aspects, the serial-flow resistance trap can be paired with amicrofluidic device. The device can consists of an inlet, where samplecan be introduced (left side), as well as an outlet, where excess liquidphases can be removed (right side). The center of the device can have ahigh density of flow resistance traps which can be incorporated. FIG.18B shows a magnified region of FIG. 18C. In some aspects, the flowresistance traps can constitute a functional part of the device.

The 3D device can have chambers with a variety of potential dimensionsand shapes. The dimensions and shapes can affect the parallel flowresistance trap design. In some aspects, the chambers of the 3D devicecan be wells. The device can trap single particles/cells from a solution(e.g., such as beads, cells, etc.) and multiple wells can be arranged inparallel to form the device. A cross-sectional side view of a well onthe 3D device can illustrate potential shape variations (FIG. 19). Insome aspects, the well design can include a helix, ellipse, parabola,hyperbola, polygon, apeirogon, chiliagon, decagon, enneagon, googolgon,hectagon, heptagon, hendecagon, hexagon, megagon, myraigon, octagon,pentagon, quadrilateral, triangle, trapezium, cylinder, hyperplane,plane, platonic solid, dodecahedron, hexahedron, icosahedrons,octahedron, tetrahedron, torus, quadric, done, cylinder, sphere,hyperboloid, paraboloid, polychoron, hecatonicosachoron, hexacosichoron,hexadecachoron, icositetrachoron, pentachoron, tesseract, sphericalcone, or dome. In some aspects, the dimensions of the well can bedefined by h₁=depth of well, h₂=depth of constriction, r₁=radius ofwell, and r₂=radius of constriction.

In some aspects, the physical material of the device can consist of anoptically transparent material, including, but not limited to glass,polymer (e.g., PDMS, mylar, Dymax, polyurethane). In other cases, thephysical material of the device can be constructed from multiple layers.In some aspects, the multiple layers can comprise various layers of theaforementioned materials.

The device described in the disclosure can be built using methodsdescribed herein. For example, FIG. 20 is a schematic depiction of amethod that can be used to build some of the devices. In some aspects,the method can include building the device layer by layer. For the firstlayer, a solid substrate (e.g., silicon wafer) can be spin-coated withphotoresist. The coated substrate can be placed in hard contact with aphotolithographic mask imprinted with the desired design depicted inUV-transparent and opaque regions. The mask and substrate can be exposedto UV-light. The UV-light can be used to crosslink photochemicals in thephotoresist. The first layer of the fabricated device can be completedby developing the resultant crosslinked pattern, dissolving away thenon-crosslinked portion of the photoresist. In some aspects, the secondlayer can be built using a mold. The mold can be prepared by coating,exposing, and developing another layer of photoresist. In some aspects,the mold can be used for forming channel, chamber, and well structuresin a curable (e.g., PDMS) or embossable material. For example, the moldcan be placed into a dish. In some aspects, the PDMS can be poured overthe mold and cured. The PDMS can be released from the mold, and inletsand outlets can be punched. In some aspects, the patterned PDMS can besealed to a flat glass or piece of PDMS to enclose the channels andchambers or wells.

The device described in the disclosure can be built using methodsdescribed herein. For example, a microfabrication method can be used toproduce the parallel flow resistance trap (FIG. 21). The device can befabricated in material other than photoresist (e.g., SU-8), which caninclude, but is not limited to, polymeric material, photoresist,polymethydisoloxane (PDMS), polymethylmethacrylate (PMMA),polymethylurethane (PUMA), etc. In some aspects, the microfabricationmethod can include, (1) spin coating a sacrificial layer on a silicon(Si) wafer, (2) depositing photoresist (SU-8) on top, (3) aligning aphotomask with the microarray pattern and exposing the wafer to UV, (4)processing and removing the uncrosslinked photoresist, if needed, spincoating and processing a second layer of photoresist, and finally (5)releasing the SU-8 layer from the Si wafer and assembling onto (path 1only) a porous polycarbonate filter and/or (path 1-2) a PDMS mount witha single outlet for inserting tubing.

In some aspects, single cells can be trapped by fluid forces. The fluidforces can hold the cells. In some aspects, the fluid forces can beenhanced by interaction with molecules on the surface of the device. Insome aspects, molecules can exhibit specific binding (e.g., aptamers,antibodies, primary amines, succinymidyl esters) or non-specific binding(e.g., polyelectrolytes). In other cases, the fluid forces can beenhanced by optical forces. The optical forces can include but are notlimited to, attractive forces (e.g., single beam gradient optical trap,vortex trap), repulsive forces (e.g., scattering), interfacial forces(e.g., Marangoni forces), electrostatic forces (repulsive forces,attractive forces) or by magnetic forces (e.g., exerted by theinteraction of applied magnetic fields with inherent magneticallysusceptible internal species).

The disclosure provides a device, method, and system that can performsequestration or trapping, manipulation, and detection of a sample. Insome aspects, the samples can consist of analytes being trapped (e.g.,held in a fixed position by forces generated from fluid flow, gravity,optical forces, or molecular interactions or formation of covalentbonds) in a microfluidic device.

In some aspects, an analyte is trapped at, immobilized at, held at, orotherwise attached to a micro-cavity, such as a well or compartment in amicrofluidic device. In further aspects, the analyte is within themicro-cavity. In some aspects, an analyte is trapped at, immobilized at,held at, or otherwise attached to a micro-patch, such as a patch coatedwith a substrate in a microfluidic device. In further aspects, theanalyte is trapped at, immobilized at, held at, or otherwise attached toa micro-cavity or micro-patch through forces generated from fluid flowand/or gravity. In some aspects an analyte is trapped at, immobilizedat, held at, or otherwise attached to a micro-cavity or micro-patchthrough an adhesive force, a molecular interaction and/or a covalentbond. In some aspects an analyte is trapped at, immobilized at, held at,or otherwise attached to a micro-cavity or micro-patch through anon-covalent bond, including without limitation a van der Waalsinteraction, an electrostatic interaction, a hydrophobic interactionand/or a non-specific adsorption.

In some aspects, the analytes are single cells. The analytes can becontacted with a tag. In some aspects, the tag can recognize a target onthe analyte. The tag can bind to the target. In some aspects, the targetcan be a molecular component of the analyte. In some aspects themolecular component can be exterior to the analyte. In other cases, themolecular component can be interior to the analyte. The tags can bedetected using a variety of detection methods.

The disclosure provides a method for containment or physical trapping ofsingle analytes. In some aspects, the analytes can be cells. The cellscan be transported in a liquid phase where the liquid can follow theflow path and can transit to a physically defined position. In someaspects, the cells can remain in the defined position. The flow basedforces can be placed upon the cell to trap the cell in the definedposition. In another case, the cells can be trapped sequentially as thefluidic flow path is serial with respect of inlet to outlet. In yetanother case, multiple fluid flowpaths and their commensurate multiplesingle cells can be trapped or sequestered in a parallel manner. In someaspects, trapping in a parallel manner can be due to the numerous flowpaths that can simultaneously be experienced by the cells between theinlet and outlet.

In a preferred case, the method can provide for each analyte (e.g.,cell) in a fluid sample to follow a fluid path and enter a chamber. Theanalyte can enter the chamber and block the fluid path stopping the flowthrough the fluid path. In this case, another analyte may not enter thechamber. The remaining cells in the fluid sample can enter the remainingempty chambers until some, or all, of the chambers contain an analyte.

In some aspects, the method by which the serial-flow resistance trapfunctions to collect, discretize, and readout biologically derivedsamples is disclosed herein. For example, FIG. 22A shows how samples canserially fill defined locations. This process can occur when thecritical dimension (diameter) of the sample is, (1) smaller than theheight and width of the main channel, and (2) larger than the height orwidth of the constriction. In some aspects, differential resistance toflow can direct the sample into the defined region, whereupon theconstriction can be occluded and flow can be stopped. Subsequent samplescan follow a flow path through the main channel, to the next trappinglocation, until all traps are filled. For example, FIG. 22B shows how animmiscible liquid phase can be introduced through the sample inlet. Theimmiscible liquid can serially discretizes the trapped samples. In someaspects, the immiscible liquid may not flow into the sample regions. Thelack of fluid flow of the immiscible liquid can be attributed to theoccluded restriction. In some aspects, the lack of fluid flow can beattributed to an interfacial barrier created by the differential contactangles of the immiscible phases with the channel material and thedimensions of the trapping region. In some aspects, temporal informationregarding each sample can be physically encoded in each location. Thevarying chemical natures of the discretized samples can be detected(FIG. 22C).

In some aspects, the method by which the parallel flow resistance trapfunctions to collect, discretize, and readout biologically derivedsamples is disclosed herein. For example, FIGS. 22A and 22D show howsamples can fill the parallel flow resistance trap. In some aspects,sample particles can follow the flow from the top of the device into thewells. Multiple samples can be trapped simultaneously in the parallelflow resistance trap. In some aspects, the speed of sample collectioncan be faster using the parallel flow resistance trap rather than theserial-flow resistance trap. For example, FIG. 22D shows how animmiscible phase can be replaced above and below the plane of thetrapping wells. In some aspects, the immiscible phase above and belowthe plane of the trapping wells can generate the discretized samples. Insome aspects, temporal information regarding each sample can bephysically encoded in each location. The varying chemical natures of thediscretized samples can be detected (FIG. 22C).

In some aspects, the trapped analyte can be released from thesingle-analyte array and analyzed. For example, FIG. 24 illustrates apossible sequence for trapping an array of biological analytes foranalysis and release. In some aspects, the sequence can include, (1)applying flow from the tubing to move the fluid flow through the trap,(2) ceasing the flow in the localized region once analytes are trapped,and, (3) rinsing excess fluid and particles using a buffer.

In some aspects, the analytes trapped in the single-analyte array canundergo further analysis. In some aspects, the single-analyte array canbe used with the method of sequential immunostaining and photobleaching.

In some aspects, the single-analyte analyte array can be used withmethods known to those of skill in the art for analysis of nucleicacids. In some aspects, the analysis of nucleic acids can includepolymerase chain reaction (PCR). In the case of performing PCR using thesingle-analyte array (FIG. 17), all the chambers can be filled with asingle analyte in a fluid. Each chamber can be sealed (e.g., using oil)and PCR can be performed on each single analyte in parallel.

Analytes contained in the individual wells or chambers of thesingle-analyte array can be detected using a variety of techniques. Forexample, the analytes in the wells or chambers can be imaged usingmicroscopy. In some aspects, microscopy can include bright fieldmicroscopy. In some aspects, microscopy can include fluorescencemicroscopy. In any case of microscopy, the sample can be placed on astage. In some aspects, the stage can be manually operated. In othercases, the stage can be an automated translation stage that can becontrolled by computer programs. In any case of microscopy, images canbe acquired by CCD cameras.

Applications

The disclosure provides for the method to be used in a variety ofapplications. In some aspects, the method can provide a subject adiagnosis or prognosis for a condition associated with the presence of arare particle in a fluid sample, for example a biological fluid such asa blood sample.

The methods and apparatuses described herein can comprise the steps of:(a) contacting a biological fluid from the subject with a tag underconditions suitable to transform the tag into a complex comprising saidtag and an analyte; (b) detecting the presence or absence of a complexformed in step (a) in an aliquot of the biological fluid; (c) assigninga value to the aliquot based on the presence or absence of a complexformed in step (a); and (d) providing a diagnosis or prognosis to thesubject based on analysis of the isolated aliquot. In some aspects, step(b) can comprise interrogating the aliquot with a source of radiationand detecting a signal emitted by the tag bound to the analyte.

In various aspects, methods are provided for identifying a plurality ofmarkers present on an analyte within a fluid, wherein the methodcomprises: (a) detecting a signal from a first tag using a source ofradiation, wherein the first tag is attached to a first structure thatbinds to a first marker on the analyte; (b) partitioning the analytebased on the presence of the first tag; (c) reducing the intensity ofthe signal of the first tag; (d) contacting the analyte with a secondstructure that binds to a second marker, wherein the second structure isattached to a second tag; and (e) detecting the second tag.

In some aspects, the partitioning of step (b) is based on the presenceof the first tag and a third tag. In further aspects, the partitioningof step (b) is performed with a microfluidic device. In still furtheraspects, the partitioning of step (b) and the detecting in at least oneof step (b) or step (e) occurs within the same microfluidic device.

In various aspects, methods are provided for detecting a plurality ofmarkers present on an analyte, the method comprising: contacting theanalyte with a first tag, wherein the analyte comprises a first markerand the first tag has an affinity for the first marker; detecting afirst signal emitted by the first tag, wherein the presence of the firstsignal indicates the presence of the first marker; partitioning a fluidcomprising the analyte based on the presence of the first signal;reducing the intensity of the first signal; contacting the analyte witha second tag, wherein the analyte comprises a second marker and thesecond tag has an affinity for the second marker; and detecting a secondsignal emitted by the second tag, wherein the presence of the secondsignal indicates the presence of the second marker.

In some aspects, the signal of the first tag is reduced by greater than50%. In certain aspects, reducing the intensity of the signal isaccomplished by applying radiation to the analyte. In further aspects,white light is used to reduce the intensity of the signal. In stillfurther aspects, a laser is used to reduce the intensity of the signal.In other aspects, a light emitting diode is used to reduce the intensityof the signal. In some aspects, reducing the intensity of the signal isaccomplished by applying a chemical to the first tag. In certainaspects, the chemical is a reducing agent. In further aspects, thereducing agent is dithiothreitol.

In some aspects, partitioning the fluid is based on the presence of thefirst signal and a third signal. In further aspects, detecting the firstsignal, detecting the second signal, partitioning the fluid, or acombination thereof is performed using a microfluidic device.

In some aspects, the analyte is a cell. In certain aspects, the cell isa cancerous cell. In further aspects, the cancerous cell is a rare cell.In some aspects, the analyte is a circulating tumor cell. In certainaspects, the cell is a bacterial cell, an immune cell, a fetal cell, acell indicative of a disease remaining after treatment, or a stem cell.In further aspects, the cell is a rare cell, comprising less than 1% ofthe total cell population in the fluid. In certain aspects, the analyteis a dissociated cell.

In some aspects, the fluid is selected from the group consisting of:whole blood, fractionated blood, serum, plasma, sweat, tears, ear flow,sputum, lymph, bone marrow suspension, lymph, urine, saliva, semen,vaginal flow, feces, transcervical lavage, cerebrospinal fluid, brainfluid, ascites, breast milk, vitreous humor, aqueous humor, sebum,endolympth, peritoneal fluid, pleural fluid, cerumen, epicardial fluid,and secretions of the respiratory, intestinal and genitourinary tracts.In certain aspects, the fluid is whole blood. In other aspects, thefluid is fractionated whole blood.

In some aspects, the first tag is a fluorophore. In further aspects, thefirst tag is an antibody. In other aspects, the first tag is a probecomprised of a nucleic acid. In certain aspects, the methods furthercomprise imaging the signal from the first tag and the second tag. Insome aspects, the analyte is present in a fluid, and the fluid is analiquot of a larger volume of fluid. In further aspects, detecting thefirst signal emitted by the first tag is performed simultaneously on aplurality of analytes.

In certain aspects, the partitioning is performed semi-automatically orautomatically. In certain aspects, the partitioning is performed byensemble-decision aliquot ranking. In some aspects, a flow cytometer isnot used to partition the analyte.

In further aspects, the analyte comprises a plurality of markers. Insome aspects, each of the plurality of markers is a biomarker. Incertain aspects, the analyte comprises an expression profile, whereinthe expression profile is defined by the plurality of markers.

In further aspects, the method further comprises contacting the analytewith a buffer. In still further some aspects, the buffer contains afixative. In certain aspects, the buffer contains a permeabilizationagent. In other aspects, the buffer is a washing buffer.

In various aspects, methods are provided for isolating cells from asample comprising a first cell type and a second cell type, the methodscomprising: (a) introducing the sample into a microfluidic chip via aset of tubing wherein the microfluidic chip comprises (i) at least onechannel fluidly connected to the set of tubing; (ii) a detectorconfigured to detect signals of cells within the at least one channel;and (iii) at least one chamber fluidly connected to the at least onechannel; (b) flowing a portion of the sample past the detector; (c)using the detector to detect the presence or absence of the first celltype within the portion of the sample; (d) if the first cell type isdetected within the portion of the sample, directing an aliquot of thesample into the chamber, wherein the aliquot comprises the first celltype; and (e) repeating steps (b), (c), and (d), thereby isolatingmultiple aliquots in the chamber such that the chamber comprises greaterthan 80% of a total number of first cell types within the sample andless than 5% of a total number of second cell types within the sample.

In various aspects, methods are provided for isolating cells from asample, the methods comprising: (a) introducing the sample into amicrofluidic chip, wherein the sample comprises a first cell type and asecond cell type, and wherein the microfluidic chip comprises: achannel; a detector configured to detect a signal emitted within thechannel; a chamber in fluidic communication with the channel; (b)flowing a portion of the sample through the channel, wherein the portioncomprises a plurality of the first cell type, a plurality of the secondcell type, or a combination thereof; (c) detecting the presence orabsence of the first cell type within the portion using the detector;(d) directing the portion into the chamber if the first cell type ispresent within the portion; and (e) repeating (b), (c), and (d) asufficient number of times such that the chamber comprises more than 80%of the total number of the first cell type present within the sample andless than 5% of the total number of the second cell type present withinthe sample.

In some aspects, at least one of the first cell type and the second celltype is a cancerous cell. In certain aspects, the cancerous cell is arare cell. In further aspects, at least one of the first cell type andthe second cell type is a circulating tumor cell. In still furtheraspects, at least one of the first cell type and the second cell type isa bacterial cell, an immune cell, a fetal cell, a cell indicative of adisease remaining after treatment, or a stem cell. In some aspects, atleast one of the first cell type and the second cell type is a rarecell, comprising less than 1% of the total cell population in thesample.

In some aspects, the sample is selected from the group consisting of:whole blood, fractionated blood, serum, plasma, sweat, tears, ear flow,sputum, lymph, bone marrow suspension, lymph, urine, saliva, semen,vaginal flow, feces, transcervical lavage, cerebrospinal fluid, brainfluid, ascites, breast milk, vitreous humor, aqueous humor, sebum,endolympth, peritoneal fluid, pleural fluid, cerumen, epicardial fluid,and secretions of the respiratory, intestinal and genitourinary tracts.In certain aspects, the sample is whole blood. In other aspects, thesample is fractionated whole blood.

In some aspects, the chamber is external to the microfluidic chip. Incertain aspects, wherein the chamber is a vial, a microcentrifuge tube,or a well in a well plate. In further aspects, the chamber is in fluidiccommunication with the channel via tubing. In still further aspects, thechamber is in fluidic communication with the channel via a capillarytube.

In various aspects, methods are provided for identifying a plurality ofmarkers present on an analyte, wherein the methods comprise: (a)partitioning a plurality of analytes by flowing the analytes over asubstrate comprising a plurality of micro-cavities or micro-patches,wherein the majority of micro-cavities or micro-patches are capable ofcontaining not more than one analyte and wherein the micro-cavities ormicro-patches are located in a microfluidic device; (b) in themicro-cavities or micro-patches, contacting each analyte with a firststructure that is capable of binding to a first marker, wherein thefirst structure is connected to a first tag; (c) detecting a signal fromthe first tag; (d) reducing the level of the signal of the first tag;(e) contacting the analyte with a second structure that binds to asecond marker, wherein the second structure is connected to a secondtag; and (f) detecting the second tag.

In some aspects, the contacting of step (b) is achieved by flowing afluid comprising the first structure through a channel that is in fluidcommunication with the micro-cavities or micro-patch. In certainaspects, following the contacting step of step (b), the method furthercomprises: contacting the analyte with a wash buffer.

In various aspects, methods are provided for detecting a plurality ofmarkers present on an analyte, the methods comprising: isolating ananalyte in a micro-cavity or in a micro-patch by flowing a fluid over asubstrate comprising the micro-cavity or micro-patch, wherein the fluidcomprises the analyte; contacting the analyte with a first tag, whereinthe analyte comprises a first marker, and wherein the first tag has anaffinity for the first marker; detecting a first signal emitted by thefirst tag, wherein the presence of the first signal indicates thepresence of the first marker; reducing the intensity of the firstsignal; contacting the analyte with a second tag, wherein the analytecomprises a second marker, and wherein the second tag has an affinityfor the second marker; and detecting a second signal emitted by thesecond tag, wherein the presence of the second signal indicates thepresence of the second marker.

In some aspects, the method is performed on a plurality of analytesisolated in a plurality of micro-cavities or micro-patches. In certainaspects, the signal of the first tag is reduced by greater than 50%. Infurther aspects, at least one analyte is a cell.

In some aspects, an analyte is held in a fixed position within themicro-cavities by a force generated by fluid flow, gravity, or adhesiveforces. In certain aspects, an analyte is connected to a micro-cavity ormicro-patch through a molecular interaction. In further aspects, ananalyte is connected to a micro-cavity through a non-covalent bond. Instill further aspects, the non-covalent bond is a van der Waalsinteraction, an electrostatic bond, a hydrophobic bond or a non-specificadsorption.

In various aspects, methods are provided for isolating an aliquot of afluid sample within a microfluidic chip, wherein the aliquot comprises arare particle, the methods comprising the steps of: (a) detecting thepresence or absence of the rare particle in the aliquot; (b) assigning avalue to the aliquot based on the presence or absence of the rareparticle; and (c) directing the flow of the aliquot based on theassigned value by opening an electro-actuated valve, wherein theelectro-actuated valve is located on a device that is external to themicrofluidic chip. In some aspects, the microfluidic chip comprises asample input channel, at least two output channels, and at least onedirectional flow channel, and wherein the electro-actuated valvecontrols the flow of fluid within the directional flow channel.

Diagnosis of Disease

In some aspects, analytes (e.g., cells) isolated using the presentmethod can be further subjected to subpopulation analysis (e.g.,according to genotype or phenotype) to develop a targeted treatment. Forexample, the isolated cells (e.g., tumor cells) can be incubated withtags (e.g., fluorescent antibodies) binding to specific biomarkers(e.g., drug targets) to determine the presence or degree of expressionof a drug target. Once the expression of the drug target is confirmed,drugs can be chosen for therapy that are specifically developed totarget the expression of a particular drug target. In one example, theisolated tumor cells can be incubated with fluorescent antibodiesbinding specifically to Her2 receptor to determine whether the breasttumor shedding CTCs is Her2-positive. If the isolated tumor cellsexhibit high Her2 expression, Herceptin (trastuzumab) can be used as atherapy as this drug is designed to target and block the function ofHER2 protein overexpression. Other known drug targets, including BCR-ABLor PDGFR (targeted by drug Gleevec), ERBB2 (targeted by Herceptin), EFGR(targeted by Iressa, Tarceva), RAR-alpha (targeted by ATRA), Oestrogenreceptor (targeted by Tamoxifen), aromatase (targeted by Letrazole),androgen receptor (targeted by Flutamide, Biclutamide), CD20 (targetedby Rituximab), VEGF-receptor (targeted by Avastin) can also be similarlyscreened from the isolated tumor cells before prescribing theappropriate chemotherapy regimen.

In some aspects, the analyte can be a rare particle (e.g., a cancer cellor circulating tumor cell (CTC)). In other cases, the rare particle canbe a parasitic cell or organism, for example, a species of Giardia orCryptosporidium, a erythrocyte infected with a species of Plasmodium, alymphocyte or leucocyte infected with HIV, a fetal cell in maternalblood, a stem cell, a prion-infected cell, a CD4+ T-cell, and the like.

In some aspects, the method can comprise detecting a circulating tumorcell in a blood sample from a subject. The method can include eDAR. Incertain aspects, the subject can be a patient who has been diagnosedwith cancer. In some aspects, the cancer can be Stage I, Stage II, StageIII, or Stage IV. In some aspects, circulating tumor cells (CTCs) can bedetected in a blood sample from a patient previously diagnosed withcancer. In these cases, the patient can be further diagnosed withmetastatic cancer.

In some aspects, the method can be used for diagnosing metastatic cancerin a subject that has previously been diagnosed with a solid tumor, themethod comprising the steps of: (a) detecting the presence or absence ofa CTC in an aliquot of a blood sample from the subject; (b) assigning avalue to the aliquot based on the presence or absence of the CTC; and(c) directing the flow or collection of the aliquot based on theassigned value.

In some aspects, the absence of CTCs in the blood sample can becorrelated with the subject not having metastatic cancer. The presenceof at least one CTC in the blood sample can be correlated with thesubject having metastatic cancer. In some aspects, the presence of atleast a number of CTCs in the blood can be correlated with the subjecthaving metastatic cancer. The method can further comprise a step of (d)diagnosing the subject as not having metastatic cancer if no CTCs aredetected in the blood sample or diagnosing the subject as havingmetastatic cancer if at least one CTC is detected in the blood sample.

The disclosure also provides for a method for monitoring a subjectdiagnosed with cancer. The method can comprise detecting the presence orabsence of a CTC in an aliquot of a blood sample from the subject usingthe eDAR method provided herein. In some aspects, the patient can bemonitored for the progression of cancer to metastatic cancer at regularintervals, for example, at least once a year, at least twice a year, atleast 3, 4, 5, 6, 7, 8, 9, 10, or more times a year, or at least about3, 4, 5, 6, 7, 8, 9, 10, or more times a year. In some aspects, thesubject can be monitored once a month, or at least 2, 3, 4, 5, 6, 7, 8,9, 10, or more times a month. In some aspects, the subject can bemonitored about once a month, or at least about 2, 3, 4, 5, 6, 7, 8, 9,10, or more times a month. The absence of CTCs in the blood sample canbe correlated with the subject not having metastatic cancer. Thepresence of at least one CTC in the blood sample can be correlated withthe subject having metastatic cancer. The presence of at least a numberof CTCs in the blood can be correlated with the subject havingmetastatic cancer.

In cases wherein a CTC is detected in a blood sample, the method canfurther comprise a step of subjecting one or more aliquots identified ascontaining a CTC to further analysis to identify one or morecharacteristics of the CTC cell or cells. For example, an aliquot orpool of aliquots containing a CTC can be contacted with one or moredetection reagents specific for one or more cancer-specific surfaceantigens. Non-limiting examples of cancer-specific surface antigens thatcan be assayed for include, without limitation, BCR-ABL or PDGFR(targeted by drug Gleevec), ERBB2 (targeted by Herceptin), EFGR(targeted by Iressa, Tarceva), RAR-alpha (targeted by ATRA), Oestrogenreceptor (targeted by Tamoxifen), aromatase (targeted by Letrazole),androgen receptor (targeted by Flutamide, Biclutamide), CD20 (targetedby Rituximab), VEGF-receptor (targeted by Avastin), and the like. Insome aspects, the surface antigens can be assayed using the sequentialimmunostaining and photobleaching method described herein.

The methods and devices provided herein can be used to diagnose diseasesother than cancer. In some aspects, malaria can be diagnosed, the methodcomprising detecting an erythrocyte infected with Plasmodium. In anothercase, a method for diagnosing an HIV infection is provided, the methodcomprising detecting a lymphocyte or leucocyte infected with the HIVvirus using an eDAR method and/or apparatus provided herein. In yetanother case, a method for diagnosing a disease associated with a prionis provided, the method comprising detecting a prior in a biologicalfluid from a human or other animal.

In some aspects, the analyte is a rare cell and the rare cell is abacterial cell. In some aspects, the bacterial cell is present in awhole blood sample. In other aspects, a device, method, system orapparatus is used to detect the presence of the rare bacterial cell inwhole blood. In some aspects a device, method, system or apparatus isused to detect the presence of a rare bacterial cell in a whole bloodsample in order to provide a diagnosis, prognosis, or other therapeuticparameter related to characterizing or managing a bacterial infection orsepsis.

Prognosis of Disease

The present disclosure provides methods for a prognosis for a disease orcondition associated with the presence of an analyte in a fluid. In someaspects, the analyte can be a biological particle. In some aspects thefluid can be a biological fluid. In some aspects, the method comprisesthe steps of: (a) detecting the presence or absence of the analyte in analiquot of a sample from a subject; (b) assigning a value to the aliquotbased on the presence or absence of the analyte; (c) directing the flowor collection of the aliquot based on the assigned value; and (d)providing either a good prognosis if no analytes are detected in thesample or a poor prognosis if analytes are detected in the sample.

In some aspects, the aliquot can be assigned a value based on thequantity or the identity of the analytes in the aliquot. In someaspects, a good or poor prognosis can be provided based on the quantityof the analytes in the sample. For example, a good prognosis can beprovided if the quantity of the analytes in the sample is less than apredetermined reference value and a poor prognosis is provided if thequantity of the analytes in the sample is equal to or greater than thereference value. In some aspects, a predetermined reference value can beassociated with a likelihood of responding to a particular therapy or alikelihood of overall or disease free survival for a period of time, forexample at least 6 month, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, or more years.

In some aspects, a method provided herein can be used to provide aprognosis for any disease associated with a rare particle. In someaspects, a method for providing a prognosis for malaria is provided, themethod can comprise determining the number of erythrocytes infected withPlasmodium in a blood sample from an individual using an eDAR methodand/or apparatus provided herein. In some aspects, either a goodprognosis if the total number of infected erythrocytes detected in theblood sample is less than a predetermined reference value or a poorprognosis if the total number of infected erythrocytes detected in thesample is equal to or greater than the reference value can be provided.In some aspects, a method for providing a prognosis for an HIV infectionis provided, the method can comprise determining the number oflymphocytes or leucocytes infected with an HIV virus in a blood samplefrom an individual using an eDAR method and/or apparatus provided hereinand providing either a good prognosis if the total number of infectedcells detected in the blood sample is less than a predeterminedreference value or a poor prognosis if the total number of infectedcells detected in the sample is equal to or greater than the referencevalue. In some aspects, a method for providing a prognosis for a diseaseassociated with a prion is provided, the method can comprise determiningthe number of prions in a biological fluid sample from a subject usingan eDAR method and/or apparatus provided herein and providing either agood prognosis if the total number of prions detected in the sample isless than a predetermined reference value or a poor prognosis if thetotal number of prions detected in the sample is equal to or greaterthan the reference value.

In some aspects, the present disclosure provides a method for providinga prognosis for a subject diagnosed with a solid tumor. In some aspects,the method comprises the steps of (a) detecting the presence or absenceof a CTC in an aliquot of a blood sample from the subject; (b) assigninga value to the aliquot based on the presence or absence of the CTC; (c)directing the flow or collection of the aliquot based on the assignedvalue; and (d) providing either a good prognosis if no CTCs are detectedor a poor prognosis if a CTC is detected.

In some aspects, a method is provided for providing a prognosis for asubject diagnosed with metastatic cancer. In some aspects the methodcomprises the steps of (a) detecting the presence or absence of a CTC inan aliquot of a blood sample from the subject; (b) assigning a value tothe aliquot based on the number CTCs detected in the aliquot; (c)directing the flow or collection of the aliquot based on the assignedvalue; and (d) providing either a good prognosis if the total number ofCTCs detected in the blood sample is less than a predetermined referencevalue or a poor prognosis if the total number of CTCs detected in thesample is equal to or greater than the reference value.

In some aspects, a predetermined reference value can be associated witha likelihood of responding to a particular therapy or a likelihood ofoverall or disease free survival for a period of time, for example atleast 6 month, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, ormore years.

Response of Disease to Treatment

The present disclosure provides methods for monitoring the progressionof a disease or the response to a therapy. In some aspects, the methodcan comprise detecting an analyte in a fluid sample. In some aspects,the method comprises the steps of: (a) detecting the presence or absenceof the analyte in a plurality of aliquots of a first biological sampletaken from a subject at a first time; (b) assigning a value to thealiquots based on the presence, absence, quantity, or identity of therare particle; (c) determining the total value of all the aliquots fromthe first sample; (d) detecting the presence or absence of the analytein a plurality of aliquots of a second biological sample taken from thesubject at a second time; (e) assigning a value to the aliquots based onthe presence, absence, quantity, or identity of the analyte; (f)determining the total value of all the aliquots from the second sample;and (g) comparing the total value assigned to the first sample to thetotal value assigned to the second sample, wherein an increased valueassigned to the second sample as compared to the first sample iscorrelated with a progression of the disease and/or a poor response tothe therapy and/or a decreased value assigned to the second sample ascompared to the first sample is correlated with a regression of thedisease and/or a good response to the therapy. In some aspects, thealiquots can further be directed into a particular channel or chamber(channeled) based on the value assigned for collection, furtherenrichment, or further analysis.

In some aspects, methods of monitoring disease progression or responseto therapy can be employed on a regular basis after diagnosis of thedisease or initiation of the treatment regime. For example, samples canbe collected from a subject at least once a year, at least twice a year,at least 3, 4, 5, 6, 7, 8, 9, 10, or more times a year, or at leastabout 3, 4, 5, 6, 7, 8, 9, 10, or more times a year. In some aspects,the subject can be monitored once a month, or at least 2, 3, 4, 5, 6, 7,8, 9, 10, or more times a month. In some aspects, the subject can bemonitored about once a month, or at least about 2, 3, 4, 5, 6, 7, 8, 9,10, or more times a month.

In some aspects, wherein a progression of the disease or poor responseto a therapy is found, the method can further comprise a step ofassigning a therapy, increasing a dosage regime, changing a therapeuticregime, and the like. In some aspects, the disease or conditionassociated with a rare particle can be cancer, malaria, HIV/Aids, aprion-related disease, or the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. The term “about” as used herein refers to a rangethat is 15% plus or minus from a stated numerical value within thecontext of the particular usage. For example, about 10 would include arange from 8.5 to 11.5.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention may be embodied in practice. Thefollowing definitions and explanations are meant and intended to becontrolling in any future construction unless clearly and unambiguouslymodified in the following examples or when application of the meaningrenders any construction meaningless or essentially meaningless. Incases where the construction of the term would render it meaningless oressentially meaningless, the definition should be taken from Webster'sDictionary, 3rd Edition or a dictionary known to those of skill in theart, such as the Oxford Dictionary of Biochemistry and Molecular Biology(Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

As used herein and unless otherwise indicated, the terms “a” and “an”are taken to mean “one,” “at least one” or “one or more.” Unlessotherwise required by context, singular terms used herein shall includepluralities and plural terms shall include the singular.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words “comprise,” “comprising,” and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to.”

Unless otherwise specified, the presently described methods andprocesses can be performed in any order. For example, a methoddescribing steps (a), (b), and (c) can be performed with step (a) first,followed by step (b), and then step (c). Or, the method can be performedin a different order such as, for example, with step (b) first followedby step (c) and then step (a). Furthermore, those steps can be performedsimultaneously or separately unless otherwise specified withparticularity.

Words using the singular or plural number also include the plural andsingular number, respectively. Additionally, the words “herein,”“above,” and “below” and words of similar import, when used in thisapplication, shall refer to this application as a whole and not to anyparticular portions of the application.

EXAMPLES

The following examples are included to further describe some aspects ofthe present invention, and should not be used to limit the scope of theinvention.

Example 1 Ensemble-Decision Aliquot Ranking Based on MicrofluidicComponents for Large-Capacity Trapping of Circulating Tumor Cells

This example describes and validates a novel eDAR platform for isolatingparticles including cells according to an aspect of the presentdisclosure.

Two factors that determined the feature and performance of an eDARplatform are an efficient and active sorting scheme and a subsequentefficient purification (e.g., purification chamber) scheme. The presenteDAR-platform optimizes these two components. In one example, the eDARplatform can include an integrated filtration area fabricated bystandard lithography methods. The microfluidic chip can be composed oftwo layers on a silicon master and can be fabricated with one-stepreplica molding into polydimethylsiloxane (PDMS). The microfluidic chipcan be finished with bonding to a glass substrate. The entire system caninclude an active sorting scheme. The analytical performance of themicrofluidic chip and hydrodynamic switching mechanisms can be optimizedfor a particular recovery efficiency (e.g., 95%), a particular falsepositive rate (e.g., 0) and a particular throughput (e.g., 4.8 mL ofwhole blood per hour).

The Microfluidic Chip

The microfluidic chip used in this example had two functional areasintegrated in the same design, the eDAR sorting area and the filtrationunit based on slit structures. The main channel in the sorting unit,which introduced the blood into the sorting junction, had a height of 50μm and width of 150 μm; all the other 4 channels were 50 μm tall and 200μm wide. The slit-filters were 5 μm tall and 5 μm wide. The maximumnumber of slits tested was 20,000.

The silicon master was fabricated using two photolithography processes(FIGS. 5, 20 and 21). The features were designed using AutoCAD(Autodesk, San Rafael, Calif.), and written on a chrome mask (TRICRCorporation, SF, CA). Positive resist lithography and deep reactive ionetching (DRIE) were chosen for forming the first layer, the micro-filterfeature. AZ 1512 was used as a positive photoresist, which was providedby Micromanufacturing Facility (MMF) at The University of Washington.DRIE process was optimized to achieve a depth in the range of 4.5-5 μm.The second layer of the eDAR feature was fabricated using the SU-8-3050as a negative photoresist (MicroChem, Newton, Mass.), and the height ofthe feature was controlled to be 50 μm. After the master was silanizedusing tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane(Sigma-Aldrich, St. Louis, Mo.), uncured PDMS was poured onto thesilicon wafer and baked for 2 hours at 70° C. The piece of PDMS with thedesired micro-feature was peeled off the silicon master, and then bondedwith a piece of cover glass using the standard process of plasmaoxidation.

Biological and Clinical Samples

Three breast cancer cell lines, SKBr-3, MCF-7, and MDA-MB-231 cells(American Type Culture Collection (ATCC), Manassas, Va.) were used tocharacterize and optimize the eDAR system. SKBr-3 cells were cultured inMcCoy's 5, MCF-7 was cultured in Eagle's Minimum Essential Medium(EMEM), and MDA-MB-231 was cultured in Dulbecco's Modified Eagle'sMedium (DMEM) (ATCC, Manassas, Va.). All cell culture media alsocontained 2 mM L-glutamine, 10% fetal bovine serum (FBS) (ATCC,Manassas, Va.), and 50 μg/mL penicillin/streptomycin (ATCC, Manassas,Va.). Incubations were done at 37° C. with 5% CO₂ in a humidifiedenvironment. The MDA-MB-231-GFP cell line was provided by Prof. GailSonenshein at Tuffs University and cultured in the DMEM medium with 10%FBS and 1 μg/mL puromycin (Life Technologies, Carlsbad, Calif.). Controlblood from healthy donors was purchased from Plasma International Lab(Everett, Wash.); the first tube of the blood draw was discarded toprevent any possible contamination from skin cells. Whole blood sampleswere drawn from patients with metastatic pancreatic cancer based on aprotocol approved by Fred Hutchinson Cancer Research Center'sinstitutional review board. Patient samples were collected at theSeattle Cancer Care Alliance (SCCA) using Vacutainer tubes (BD, FranklinLakes, N.J.) containing EDTA as an anti-coagulant, stored at 4° C., andanalyzed within 4 hours.

Sample Preparation and eDAR Analysis

Isoton (Beckman Coulter Inc., Chino, CA) was used as the buffer for allthe experiments unless otherwise specified. For a typical eDARexperiment, 1 mL of whole blood samples was labeled with anti-EpCAMconjugated with phycoerythrin (PE) (Lot No. 515776, Abnova, Walnut,Calif.) for 30 minutes at room temperature in dark. All the labelingparameters have been optimized. The labeled samples were diluted to 14mL and then centrifuged to remove the free antibodies. The final volumewas adjusted to be the same as the initial volume. The prepared samplewas next injected to the microfluidic chip using a syringe pump. Tracesfrom fiber-coupled avalanche photodiodes (APDs) (Excelitas Technologies,Waltham, Mass.) were collected by a PCI data acquisition card (PCI 6602,National Instruments, Austin, Tex.). The sorting process of eDAR wasautomatically controlled using a home-written LabVIEW (NationalInstruments, Austin, Tex.) script and a field-programmable-gate-array(FPGA) device built in house. The hydrodynamic switching, whichcollected the sorted aliquots, was controlled by a solenoid(INKA1226212H) purchased from the Lee Company (Westbrook, Conn.).

After all the positive aliquots were collected onto the filtration area,isoton was used to quickly wash the filtration area in less than 1minute. If any cytoplasmic markers were used for the secondary labeling,4% of paraformaldehyde (PFA) was loaded into the filtration area to fixthe cells. Surfynol® 465 (Air product, Allentown, Pa.) was used topermeabilize the fixed cells. Anti-EpCAM-PE and anti-Cytokeratin-APC(Lot No. MAB5131, Abnova, Walnut, Calif.) and anti-CD45-fluoresceinisothiocyanate (FITC) (Lot No. B116314, BioLegend San Diego, Calif.,)were typically used as the antibodies for the secondary labeling.Hoechst (Life Technologies, Carlsbad, Calif.) was also used as thenuclear stain to verify the labeled target was actually nucleated cells.

The method of eDAR was as follows, the blood sample was injected intothe microfluidic chip and divided into aliquots. A line-confocaldetection scheme was then applied to rank the aliquots as “positive” or“negative,” which was determined by the labeling scheme. For example, inmany applications, blood was labeled with anti-EpCAM-PE so only analiquot that had a fluorescent signal from the particular dye conjugatedto that antibody was ranked as a positive event. An automatic feedbackscheme was applied to generate a switch of the direction of blood flow,which permitted the positive aliquot to be collected quickly. Due to thevery low concentration of CTCs, more than 99.999% of the aliquots werediscarded because of the absence of the desired fluorescence signal.Those aliquots that gave the positive fluorescence signal weretransferred to another area on the microfluidic chip to be furtherpurified and then counted. A series of downstream analyses can beperformed on the trapped cells, such as a secondary immunostaining step,a more complicated staining/bleaching process, or the manipulation andculture at the single-analyte level.

Redesigned Hydrodynamic Sorting Scheme.

Previously, the eDAR had a mechanical valve to control the activesorting step, which was fast (about 2 milliseconds response time) androbust compared to other reported switching mechanisms, such as theelectroosmotic flow or the sol-gel transformation. Although promising,some design factors of this mechanical valve scheme potentiallyconstrained the potential application of eDAR. To form the mechanicalvalve on the microfluidic chip, 3 individual structural layers wererequired the solenoid, its PDMS thread, and the microchannels on a 150μm PDMS film. This would make the microfluidic chip preparationcomplicated and time-consuming. Another shortcoming was the directcontact between the captured blood aliquots and the mechanical valve,which potentially increased the risk of the loss or damage of CTCs. Inthe present example, the solenoid was replaced with an off-chip model,which is normally closed but can be opened in 2 to 3 milliseconds when a5V DC voltage is applied. Because this in-line solenoid was not a partof the microfluidic chip, the preparation of the microfluidic device wassignificantly simplified. The solenoids can be easily connected with anymicrochannels to test hydrodynamic sorting schemes. Eight differentschemes were designed and tested (FIG. 6) to drive the fluidic switch.Because of the structure of this type of solenoid and the elastic natureof PDMS, the fluidic performance varied (Table 1), and each scheme wascharacterized for the best performance.

After characterization and optimization, the structure of the platformand the corresponding scheme of the hydrodynamic sorting were chosen foreDAR (FIG. 7). The labeled blood sample was injected into the topchannel of the microfluidic chip using a syringe pump (FIG. 7a ). Twoside channels, where buffer flowed through, were used to control theactive sorting step. There were two ports placed on the right-sidechannel, and both of them were connected to a pressurized buffer source.The normally closed solenoid was connected to the port near the sortingjunction to control the hydrodynamic switch. There were two channelsafter the sorting junction. The one on the left was used to collectpositive aliquots and deliver them to the filtration and collection areafor further purification (e.g., purification chamber); the one on theright was the waste collection channel where all the negative aliquotsflowed through.

When those aliquots were ranked as “negative” (FIG. 7b ), there was novoltage applied on the solenoid so it was closed. An initial pressuredrop was set between the No. 1 and 3 buffer sources so the blood couldonly flow into the channel that collected the waste, which is also shownin the bright field image in FIG. 7b . When a positive event wasdetected by the first detection window, a 5V DC voltage was immediatelyapplied on the solenoid to open the buffer flow from the No. 2 bufferreservoir. This decreased the flow resistance of the buffer channel onthe right side and generated a higher flow rate there. The blood flowwas pushed from the right side to the left to collect the positivealiquot (FIG. 7c ). After this aliquot was collected and confirmed bythe second detection window, the solenoid was closed to switch the bloodflow back to the waste collection channel (FIG. 7d ). The time requiredfor the switch-over and back was determined to be 2 to 3 millisecondsfor each (FIG. 8). This process was stable enough for eDAR even aftermore than 10⁵ on-off cycles tested. The in-line solenoid was placed onthe buffer line so blood could not come into contact with the solenoid,which eliminated the possibility of the blood-coagulation andcross-contamination. Moreover, in this scheme, there was a constant flowof buffer in the CTC collection channel during the eDAR process. Thisimproved the efficiency of the subsequent purification (e.g.,purification chamber) step and prevented the formation of aggregates ofcells.

In some aspects of the present example, positive aliquots can becollected onto the filtration area and Isoton used to wash thefiltration area in less than 1 minute. Cytoplasmic markers can be usedfor secondary labeling and can involve loading of 4% paraformaldehydeinto the filtration area to fix the cells. Surfynol® 465 (Air product,Allentown, Pa.) can be used to permeabilize fixed cells. Anti-EpCAM-PEand anti-Cytokeratin-APC (Abnova, Walnut, Calif.) and anti-CD45−fluorescein isothiocyanate (FITC) (BioLegend San Diego, Calif.,) can beused secondary labeling. Hoechst (Life Technologies, Carlsbad, Calif.)can be used as a nuclear stain to verify nucleated cells.

Design of the Purification (e.g., Purification Chamber) Mechanism

Yet another aspect of the example can include a new scheme of on-chipfiltration based micro-slit structures that can be made of PDMS and maynot require additional layers (FIG. 10a ). FIG. 10a shows the basicstructure of an example of these microslits, which was used to capturethe CTCs without retaining any red blood cells (RBCs). The size of theslit was optimized to be 5 μm tall and 5 μm wide (FIG. 10b ), to avoidloss of any small CTCs. Many WBCs were not captured using this size offilter, which is smaller than the ones used in most of the CTC methodsbased on filtration. Because the micro-filter was made of PDMS andbonded with a piece of coverslip, the imaging quality was improvedsignificantly (FIGS. 10c and 10D) compared to the polycarbonate filter,which is not fully transparent and can generate the scattering andaberration. Moreover, because the cells could only be trapped along thearray of slits, they could be easily referenced and tracked; in manyother methods, the cells are distributed randomly on the surface. Thistrapping along the slits made the imaging procedure faster and theresults of enumeration more accurate. The slits also made it faster andmore efficient to perform the secondary labeling on the trapped CTCs.FIG. 10e shows that two cancer cells labeled with anti-EpCAM-PE weretrapped on the microslit. Cells were fixed, permeabilzed, and labeledusing anti-Cytokeratin-Alexa488, anti-Her2-Alexa647 and Hoechst.Fluorescence images showed the expression of these markers on these twocells clearly, and the bright field image also confirmed theirmorphology. Two cells were also labeled with anti-CD45-Alexa700 as anegative control marker, and no signal from the color channelcorresponded to this tag.

Characterization and Analytical Performance of eDAR

The efficiency of the active sorting step was monitored in real time.FIG. 9 shows a small portion of the APD data from a pancreatic cancerpatient sample. The “Decision APD trace” was from the first detectionwindow that ranked the aliquots and controlled the sorting. The twopeaks at 978 and 1298 milliseconds represented two CTCs labeled withanti-EpCAM-PE that triggered the aliquot sorting. The two peaks of the“Confirmation APD trace” show that two cancer cells flowed through thesecond detection window located on the collection channel, confirmingthat the two positive aliquots were actually sorted. It is worthwhile topoint out that the background change from the second detector (FIG. 9a )also confirmed that only a small portion of blood was collected by eDAR,contributing to the high enrichment ratio of CTCs (up to a million foldfor a typical clinical sample).

Because the labeled CTC had to flow from the first detection window tothe second one, a time difference between the decision APD peak and itsconfirmation signal was observed. This time difference was defined asthe transit time of the sorted CTCs, which can vary because the CTCs canhave different linear flow rates due to the nature of laminar flow inthe microchannel. FIG. 9b shows the distribution histogram of thetransit time at flow rates of 40 and 80 μL/min. Generally, a highervolumetric flow rate of the blood resulted in a shortened transit timeof the sorted CTCs (FIG. 9c ). When the flow rate was 90 μL/min, theaverage transit time was lowered to 4 milliseconds, very close to theswitching time of the sorting scheme (2 to 3 milliseconds), whichimplies that this is the limit for the throughput for this particularembodiment of a design for eDAR. In other embodiments, the present eDARdesign may yield transit times less than 2 milliseconds.

If the transit time for a sorted CTC was shorter than the hydrodynamicswitching time, the cell may not have been reliably sorted on thisplatform. The sorting efficiency was thus defined as the number ofcollected events versus the total number of events that triggered thesorting. FIG. 9d shows the values of sorting efficiency at the flow rateof 30 to 100 μL/min. When the flow rate was 30 μL/min, the sortingefficiency was almost 100% because the average transit time at that flowrate was around 10 milliseconds (FIG. 9c ). This transit time was longenough for the active sorting step to collect the CTCs. The sortingefficiency decreased to 90% at the flow rate of 80 μL/min, and thendropped to 49% when the flow rate was 90 μL/min. FIG. 9d also shows therecovery efficiency of eDAR at different flow rates, which had a similartrend compared to the sorting efficiency. However, the recoveryefficiency was defined as the number of spiked-in cells versus thenumber of recovered cells counted using multicolor fluorescence imagingon the microfluidic chip. This performance is a combination of manyfactors, including the antibody-labeling efficiency, the line-confocaldetection efficiency and the sorting efficiency. This explains thedifference between the recovery and sorting efficiency at the same flowrate. As a result, for this current generation of eDAR, the upper limitof the throughput was 80 μL/min (12.5 minutes for 1 mL of blood) with an88% recovery ratio. Although this throughput is higher than most CTCtechnologies for the analysis of whole blood, it can be further improvedby designing a wider blood inlet channel or moving the first detectionbeam farther up.

Three to 975 MCF-7 cells were spiked into 1 mL of healthy blood toanalyze the recovery efficiency at the flow rate of 50 μL/min. To ensurethe accuracy of the cell numbers at the low end, a capillary countingmethod was used to precisely spike in cultured cells when theconcentration was lower than 100 cells/mL. The average recovery ratiowas 95% with an R² value of 0.998 (FIG. 9e ), which is a little higherthan the first generation of eDAR (93%). Because the concentration ofCTCs is usually very low, the enumeration results were affected by thePoisson distribution. In this case, the ability to analyze a largervolume of whole blood sample with an acceptable throughput and recoveryratio was very important. The same number of MCF-7 cells was spiked into1, 5 and 10 mL of healthy blood, and then these three samples wereanalyzed at the flow rate of 50 μL/min. There was no significant changein their recovery ratio (FIG. 90, which shows that the method is capableof running a large amount of whole blood with high efficiency andthroughput.

Although EpCAM was used in most of the CTC studies to select tumorcells, increasingly more studies have reported that CTCs with a lowEpCAM expression have more mesenchymal characteristics and are moreaggressive. The latest eDAR platform is sufficiently flexible to use anylabeling scheme to select rare cells to capture tumor cells usingbiomarkers other than EpCAM. Three schemes were designed to selectdifferent cultured breast cancer cell lines (FIG. 9g ). EpCAM was usedto select MCF-7 cells, Her-2 was used to select SKBr-3 cells, and EGFRwas used to select MDA-MB-231 cells. All these three schemes isolatedand trapped the targeted cells with a recovery ratio higher than 88%.Another unique and important feature of eDAR is the independence ofwhere the marker is located. For example, in other technologies, such asthe surface capture methods or immunomagnetic methods, only can capturethe antigens on the cell surface. The present method was able to selectcells with an intracellular marker, such as GFP (FIG. 9d ). The recoveryratio of the MDA-MB-231-GFP cells spiked into whole human blood was 91%(FIG. 9g ). Since fluorescent proteins are widely used in animal modelsto study the progression and mechanisms of metastasis, eDAR is an idealtool to select CTCs in these models without any immunostaining steps.

High-Throughput Analysis of Samples from Patients with Pancreatic Cancer

Blood samples from 15 healthy donors were used to evaluate the falsepositive ratio of this method; no CTCs were found in any of them. 26blood samples were collected from the patients with pancreatic cancer.Sixteen of them were analyzed using the first generation of eDAR and theother 10 samples were analyzed using the newer eDAR platform. FIG. 25shows the distribution of the three data sets: the control bloodanalyzed by the current method, pancreatic cancer samples analyzed bythe first generation of eDAR, and the pancreatic cancer samples analyzedby the current method. The raw data of those clinical samples are inTable 3 (below).

TABLE 3 Shows raw data of the control and the pancreatic cancer samples.Sample Volume (mL) CTC counts Control 1 1 0 Control 2 1 0 Control 3 1 0Control 4 1 0 Control 5 1 0 Control 6 1 0 Control 7 1 0 Control 8 1 0Control 9 1 0 Control 10 1 0 Control 11 1 0 Control 12 1 0 Control 13 10 Control 14 1 0 Control 15 1 0 Patient 1 1 183 Patient 2 1 9 Patient 31 7 Patient 4 1 3 Patient 5 1 14 Patient 6 1 6 Patient 7 1 4 Patient 8 10 Patient 9 1 0 Patient 10 1 27 Patient 11 1 44 Patient 12 1 5 Patient13 1 7 Patient 14 1 8 Patient 15 1 2 Patient 16 1 10 Patient 17 1 872Patient 18 1 2 Patient 19 1 5 Patient 20 1 12 Patient 21 1 22 Patient 221 2 Patient 23 1 0 Patient 24 1 14 Patient 25 1 0 Patient 26 1 7

With this method, CTCs were detected in 80% (8 of 10) of the samplesranging from 2 to 872 cells/mL. CTC clusters, reported by previousstudies, were also observed in the patient blood samples. It isinteresting to point out that many of the clusters observed inexperiments had low EpCAM expression. FIG. 26 shows a cluster of CTCswhich had a high expression of cytokeratin but a low expression ofEpCAM.

Example 2 Imaging Multiple Biomarkers in Captured Rare Cells bySequential Immunostaining and Photobleaching

In this example, a preferred scheme for the sequential immunostainingand photobleaching process is disclosed. In a preferred example, aninline staining and washing system are coupled with eDAR in order tominimize the dead volume; decrease the amount of antibodies used; avoidintroducing air bubbles; and automate the process.

This example describes an optimized, simple and semi-automatic method toperform expression analysis of protein markers on trapped CTCs. Aninline immunostaining and photobleaching system can allow for labelingand fluorescence imaging on selected CTCs. The method can include,labeling CTCs with a group of antibodies conjugated to differentfluorophores followed by photobleaching and re-labeling with differentfluorescent antibodies against another group of biomarkers. This processcan be repeated multiple times to study several groups of proteinbiomarkers. In an exemplary case, two protein markers of interest can becombined with a positive control marker (e.g., nuclear stain) and anegative control marker (e.g., CD45) to create a group. One group can bestudied in each round, followed by photobleaching before a second round(e.g., four rounds of immunostaining and photobleaching) to look at theexpression of protein markers of interest.

Microfluidic Components and Line-Confocal Optics

The polydimethylsiloxane (PDMS) microfluidic chips were fabricated usingmethods described previously. Briefly, the features were designed usingAutoCAD (AutoDesk, San Rafael, Calif.), and then written on atransparency mask by Fineline Imaging (Colorado Springs, Colo.).Micro-features were fabricated on a silicon wafer using SU-8-3050(Micro-Chem Corp., Newton, Mass.) as a negative photoresist; the featureheight was controlled to be 50 μm. Once the features were developed,uncured PDMS was poured onto the silicon master, incubated at 75° C. for2 hours, peeled off and then bonded to a glass coverslip using theplasma oxidation method.

The line-confocal detection scheme used two laser sources, 488 and 633nm, to form the two detection windows using a series of dichroicmirrors, cylindrical lens and beam splitters. The first detectionwindow, having the two laser beams overlapped at the same time, was usedto detect the fluorescence signals from the labeled CTCs, and thencontrolled the sorting automatically. The second detection window wasused to confirm the sorted aliquots and monitor the sorting efficiency.

Biological Materials and eDAR Process

Isoton (Beckman Coulter Inc., Chino, CA) was used as the buffer for allthe experiments unless otherwise specified. The breast cancer cell linesMCF-7, SKBr-3 and MDA-MB-231 (American Type Culture Collection (ATCC),Manassas, Va.) were used to characterize the system. Cell culture wasperformed under the conditions recommended by the vendor, and harvestedonce a week. MCF-7 was cultured in Eagle's Minimum Essential Medium(EMEM); SKBr-3 cells were cultured in McCoy's 5; and MDA-MB-231 wascultured Dulbecco's Modified Eagle's Medium (DMEM) (ATCC, Manassas,Va.). All media also contained 2 mM L-glutamine, 10% fetal bovine serum(FBS) (ATCC, Manassas, Va.), and 50 μg/mL penicillin/streptomycin. Humanwhole blood drawn from healthy donors was purchased from Plasma LabInternational (Everett, Wash.) and stored at 4° C. upon arrival. Each 20mL draw came in four 5 mL Vacutainer tubes coated with EDTA as ananti-coagulant. The first tube of each draw was discarded to avoidpotential contamination from skin cells.

Antibodies were centrifuged for 5 minutes at 14,000 rpm to removepossible aggregates before any labeling procedure. Each blood sample waslabeled with anti-epithelial cell adhesion molecule (EpCAM) conjugatedwith phycoerythrin (PE) (Abnova, Taipei City, Taiwan) in darkness andincubated at room temperature for 30 min. The labeled blood sample waswashed and centrifuged (2,300 rpm for 10 min) to remove the freeantibodies. The sample was immediately injected into the microfluidicchip using a syringe pump. Typically, the flow rate was set to 50 μL/minfor the operation of eDAR, although based on the previous optimizationmethods, it can be higher. APD signal traces were collected by a PCIdata acquisition card (PCI 6602, National Instruments, Austin, Tex.) andanalyzed by a MATLAB (MathWorks, Natick, Mass.) script developedin-house. A home-built electronic box was programmed to give anautomatic feedback control based on the detected APD signals, and applya voltage on the solenoid (S-10-38-H-40, Magnetic sensor systems, VanNuys, CA) connected to the microfluidic chip. More details about eDARwere described previously.

Sequential Immunostaining and Photobleaching Process

After washing the cells isolated by eDAR, main, side and waste channelswere closed by turning off the inline valve. A 400-μL aliquot of cellfixation buffer (BioLegend, San Diego, Calif.) was introduced into themicrofluidic chip by a peristaltic pump (Fisher Scientific, Pittsburgh,Pa.) at a flow rate of 15 pt/min. After washing with the buffer for 5minutes at the same flow rate, the cells were permeablized by flowingthrough 250 μL of 2.5% surfynol 465 surfactant (Air Products andChemicals Inc, Allentown, Pa.) for 15 min. After this step, four roundsof immunostaining and photobleaching of the cells were performed. Foreach round of staining, 220 μL of a staining solution with fourbiomarkers conjugated to four different fluorescent dyes were prepared.The details about the antibodies and nuclear stain used in each roundare summarized in Table 2. After a centrifugation step (14,000 rpm for 5min) to remove the aggregates, 200 μL of the supernatant was collectedas the staining buffer. The supernatant was injected into themicrofluidic chip at a flow rate of 20 pt/min. When the antibodysolution filled the whole filtration area, the flow was stopped.Incubation took place for 20 minutes in dark to ensure all the trappedcells came into contact with the antibodies efficiently. After thisstep, the cells were washed for 10 minutes to remove any free antibodiesand minimize the fluorescence background. Photobleaching was performedusing a xenon arc lamp as the light source (Sutter instrument, Novato,Calif.). Each bleaching step took 15 min. A 20× objective was used forepi-fluorescence imaging and photobleaching. Fluorescence images werecollected before and after the photobleaching step from 4 differentemission channels: yellow (555 to 605 nm for PE), blue (435 to 485 nmfor Hoechst), green (510 to 540 nm for FITC or Alexa 488) and red (665to 695 nm for Alexa 647 or APC).

Safety Consideration for the Photobleaching Process

To ensure safety when running the photobleaching tests, the highestpower was locked to 10 mW. Certain protective methods should beconsidered when the sample is exposed to the light source, such aswearing protective goggles or covering the photobleaching area with ablack box.

CTCs Isolated by eDAR

Whole blood sample was pre-labeled with the antibodies conjugated tofluorophores and then introduced to the microfluidic chip. In manyapplications, EpCAM was used as the biomarker for the positiveselection. However, the method can be flexible in using a different ormore complicated selection logic.

In eDAR, a virtual aliquot was first defined by a combination of thelaser detection beam, the volumetric flow rate, and the sorting speed.Based on these factors, the labeled blood sample was virtually dividedup into half a million aliquots per 1 mL with 2 nL per an aliquot. Theline-confocal detection method detected the fluorescence emission withsingle-analyte sensitivity. As a result, these virtual aliquots wereranked based on the primary labeling schemes as “positive” or“negative.” Because of the very low concentration of CTCs, more than99.999% of the aliquots were discarded (FIG. 10A), which resulted in agreater than 1-million-fold enrichment ratio.

Based on the results of the aliquot ranking, an automatic feedbackmechanism was applied to trigger a hydrodynamic switch of the blood flowso that the “positive” aliquots could be collected and transferred to anarea for further purification (e.g., purification chamber) and analysis.Two design elements controlled this hydrodynamic switch—a solenoid andthe pressure drop in the two side buffer lines. A solenoid was placed inthe CTC collection channel in the closed position on the left (FIG. 4A)so the “negative” aliquots only flowed into the waste channel on theright. There was also a pressure drop between the two side channelswhere the buffer flowed, which switched the blood flow from the wastechannel to the collection side when the solenoid was open. This simplescheme was fast enough at 2 to 3 milliseconds to collect CTCs withminimum amount of blood cells. A second line-confocal detection windowwas also placed on the collection side to monitor the efficiency of thehydrodynamic switching in real time. FIG. 4B shows a small part of thedata from a sample taken from a lung cancer patient. In this figure, thegreen signal shows the APD traces from the first detection area whichcontrolled the aliquot sorting; the signal in red was the APD countsfrom the second detection area confirming that the aliquots wereactually sorted.

These sorted aliquots were transferred to an area where CTCs could betrapped and most of the blood cells discarded (FIG. 4A). Although thereare many possible ways to further purify the captured cells, a smallpiece of polycarbonate filter (5×5 μm pore size) was incorporated ontothe microfluidic chip. The trapped cells were imaged and further labeledwith more biomarkers on the microfluidic chip to determine theiridentities. For example, FIG. 26a and FIG. 26b shows a cancer celltrapped on the microfluidic chip, which was positive against EpCAM,cytokeratin, and the nuclear stain but negative against CD45. This cellwas also observed by bright-field microscopy, which provided themorphological information.

Sequential Immunostaining and Photobleaching

An inline staining and washing system was developed and coupled witheDAR in order to minimize the dead volume; decrease the amount ofantibodies used; avoid introducing air bubbles; and automate theprocess. As shown in FIG. 13A, two ports on the microfluidic chip wereleft open to perform the perfusion labeling and washing steps while allthe other three ports were completely closed. A peristaltic pumpdelivered the washing buffer and labeling reagents to the microfluidicchip and was coupled with the pressurized buffer source via a six-wayvalve. The other three ports on this valve were completely blocked toprevent any possible leakage or contamination. When running the eDARexperiment, the six-way valve was turned to the pressurized buffer sideto provide a stable control of the hydrodynamic switching. It was turnedto the peristaltic-pump side to inject accurate amounts of reagents tothe microfluidic chip without introducing any air bubbles. Using thisscheme, a few nanograms of the antibodies were introduced to the trappedcells in less than 5 min; a typical incubation step took less than 20minutes (FIG. 13B).

If there was a need to perform intracellular marker testing, thecaptured cells were fixed and permeabilized on the microfluidic chipprior to the test. Then multiple rounds of the staining, washing,imaging and bleaching experiments were performed sequentially. In eachround, four colors of fluorescence, i.e., yellow (PE), red (Alexa 647 orAPC), green (FITC), and blue (nuclear stain), were monitored.

For this part of the study, an assay for the expression of proteinmarkers on captured CTCs based on four rounds of sequentialimmunostaining and photobleaching processes was designed. Four differentmakers in each round through four individual channels usingepi-fluorescence microscopy were monitored. Each set of markers had anuclear stain (Hoechst) as a positive control marker, CD45 conjugatedwith FITC as a negative control marker, and two protein markersconjugated with PE or Alexa 647. The system was designed not to bleachthe Hoechst stain for two reasons: the stain was used as a positivecontrol marker, and it would require a UV exposure to bleach the stain,which could cause significant cellular damage. CD45 is widely expressedon many types of white blood cells (WBCs), which are considered to bethe biggest interferences in the separation of CTCs. Therefore, they arefrequently used as negative control markers.

Many protein markers can be tested on CTCs but as a proof of concept,eight antigens were selected and divided them into four groups (Table2). The first set had EpCAM and cytokeratin, which are the most widelyused markers to identify CTCs. The immunostaining test set was appliedright after the capture of CTCs by eDAR to further confirm and enumeratethe CTCs with epithelial biomarkers. Table 2 shows that there were sixtumor cells trapped on the microfluidic chip, which are positive to theHoechst stain but negative to CD45. Two of them had a strong expressionof EpCAM and cytokeratin, which implied the cells had epithelialcharacteristics.

The second set was designed to investigate other epithelial markerswhich are important for clinical and biological studies. Her2 and MUC1were selected as the two protein markers for this set since these twobiomarkers play important roles in the cancer pathogenesis andresistance to drugs. They are also potential targets of the anti-tumordrugs and immunotherapy. The second round of labeling in FIG. 14 showsthat part of the cells trapped on the microfluidic chip had MUC1expression but all of them were really low in their Her2 expression.

Cancer stem cells have been shown to play important roles in tumorprogression and have been observed in the population of CTCs. The thirdset of markers had two cancer stem cell antigens, CD44 and CD24. Theyare extensively studied as stem cell markers for breast cancer andpossibly for other types of cancers as well. The data in Table 2 showsthat 4 cells had a strong expression of CD44+/CD24−, and the other twoare CD44−/CD24+. Other stem cell markers, such as CD133 and CD105, canalso be used in this group based on the type of primary cancer.

The last set of markers in Table 2 was designed to look at theexpression of EGFR and CD166 to demonstrate the mesenchymalcharacteristics of tumor cells. EGFR has been shown to be associatedwith the EMT process, and CD166 was used to define mesenchymal stemcells in bone marrow. Other related markers, such as vimentin andcadherin, can be used in this group as well.

Characterization of Photobleaching

There are two critical factors that can determine the efficiency of thephotobleaching step—exposure power and time which were characterized andoptimized them to improve the efficiency and throughput while ensuringthat the cells were not damaged by potential heating. The photobleachingcurve under different exposure powers was studied first (FIG. 15A).MCF-7 cells were labeled with anti-EpCAM-PE, and placed on a No. 2coverslip. Labeled single cells were bleached with three different powersettings. The bleaching curves show that the exposure time can becontrolled under 10 minutes to get a more than 95% bleaching efficiencywhen the exposure power was higher than 2 mW.

Based on this, any of the bleaching curves of the four fluorophores, PE,FITC, Alexa 488 and Alexa 647, can be directly applied in the scheme.FIG. 15B shows that the fluorescent emission of PE, FITC and Alexa 488can be bleached to less than 10% in less than 5 min; the photobleachingtimes for Alexa 647 took longer, partly because the power of the lightsource between 610 to 660 nm (red excitation) was lower than that in therange of yellow and green excitation. As a result, the bleaching timewas set to 15 minutes to get a high bleaching efficiency with anacceptable throughput. This can be improved by raising the power of thelight source, although this may potentially increase the risk of heatingand cellular damage.

Example 3 Single-Analyte Trapping for Sequential Immunostaining andImaging

This example describes a single-analyte trapping apparatus coupled withthe method of sequential immunostaining and imaging.

The relevant dimensions involved in an effective serial-flow resistancetrap design, as well as a schematic of a device having arbitrarytrapping density and dimensions (FIG. 17), with an inset showing amagnified region are depicted in FIG. 18. The schematic in FIG. 18Ashows the relative dimensions of the device, such as the width of themain channel, the width of the constriction, the length from the mainchannel constriction entrance to the main channel constriction exit, thelength across the constriction, as well as the heights of the mainchannel, the constriction, and the constriction chamber. FIG. 18C showsan exemplary microfluidic device design that utilizes the serial-flowresistance trap. The design comprises an inlet where sample isintroduced (left side) and an outlet where excess liquid phases areremoved (right side). The center of the device design depicts the highdensity of flow resistance traps that is incorporated. FIG. 18B shows amagnified region of FIG. 18C and shows the flow resistance traps thatconstitute the functional part of the device.

FIG. 19 depicts a parallel flow resistance trap. Top panels of FIG. 19depict three-dimensional cross-sectional side views of wells from thedevice showing shape variations in the well and constrictions. The welldesign can be in differing or similar combinations of shapes such ascylinder, cone, square, hexagon, dome, etc. Dimensions of the well canbe defined by h₁=depth of well, h₂=depth of constriction, r₁=radius ofwell, and r₂=radius of constriction. According to this aspect, thedevice is capable of trapping single particles/cells from a solutionsuch as beads, cells, etc. Multiple wells can be arranged in parallel toform the device.

FIG. 20 is a schematic depiction of a procedure that is used to buildsome of the devices described herein this disclosure. In this process, asolid substrate (e.g., silicon wafer) is spin-coated with photoresist.The coated substrate is placed in hard contact with a photolithographicmask imprinted with the desired design depicted in UV-transparent andopaque regions. The mask and substrate are exposed to UV-light, whichinitiates photochemical crosslinking reactions in the photoresist. Thefirst layer of the fabricated device is completed by developing theresultant crosslinked pattern, dissolving away the non-crosslinkedportion of the photoresist. The second layer of the fabrication proceedsby coating, exposing, and developing a second layer of photoresist. Theresult is a mold used for forming channel, chamber, and well structuresin a curable material (e.g., PDMS) or embossable material. For example,the mold is placed into a dish. Second, PDMS is poured over the mold andcured. Third, the PDMS is released (peeled) from the mold, and at thispoint inlets and outlets are punched (not depicted). Finally, thepatterned PDMS is sealed to a flat glass or PDMS piece to enclose thechannels and chambers/wells.

FIG. 21 shows an example of a microfabrication method that is used toproduce a parallel flow resistance trap. The device is fabricated inmaterial other than photoresist (e.g., SU-8) as described above.Available materials can include but are not limited to polymericmaterial, photoresist, polymethydisoloxane (PDMS),polymethylmethacrylate (PMMA), polymethylurethane (PUMA), etc. (1) Asacrificial layer can be spin-coated on silicon (Si) wafer. (2)Photoresist (SU-8) can be deposited on top. (3) A photomask with themicroarray pattern is aligned, and the wafer is exposed to UV. (4)Uncrosslinked photoresist is processed and removed leaving the desiredpattern on the silicon wafer. A 2-layer design is used in one aspect. Inthat case, a second layer of photoresist is spin-coated and processed.(5) The SU-8 layer is released from the Si wafer and is assembled onto(path 1 only) a porous polycarbonate filter and/or (path 1-2) PDMS mountwith a single outlet for inserting tubing.

FIG. 22 depicts a set of steps by which the serial-flow resistance trapand parallel flow resistance trap function to collect, discretize, andreadout biologically derived samples. Part A shows how samples seriallyfill the defined locations. This process occurs when the criticaldimension (diameter) of the sample is smaller than the height and widthof the main channel, as well as larger than the height or width of theconstriction. Differential resistance to flow steers the sample into thedefined region, whereupon the constriction is occluded and flow isstopped. Subsequent samples follow a flow path through the main channel,to the next trapping location, until all traps are filled. Part B showshow an immiscible liquid phase is introduced through the sample inlet,and serially discretizes the trapped samples. The immiscible liquid willnot flow into the sample regions because of lack of fluid flow due tothe occluded restriction, as well as by an interfacial barrier that iscreated by the differential contact angles of the immiscible phases withthe channel material and the dimensions of the trapping region. Part Cdemonstrates how varied chemical natures of the discretized samples aredetected. As a consequence of the serial nature of filling, temporalinformation regarding each sample is also physically encoded in eachlocation. Part D illustrates the first step of samples filling theparallel flow resistance trap. Sample particles follow flow from the topof the device into the defined trapping wells. Due to the parallelnature of the design, multiple samples are trapped simultaneously forfaster sample collection than certain other trapping schemes presentedherein. Part E shows how an immiscible phase is replaced both above andbelow the plane of the trapping wells, thereby generating thediscretized samples. Part F displays how samples of varying chemicalnature are detected simultaneously.

FIG. 23 shows a detection and read-out scheme for arrays of micro-wellsand side chambers based on brightfield microscopy and fluorescencemicroscopy. The sample is placed on an automated translation stagecontrolled by computer programs. Images are acquired by high speed CCDcameras.

FIG. 24 illustrates the sequence for trapping an array of biologicalparticle/cell for analysis and release. (1) Flow is applied from thetubing to facilitate fluid flow through the trap. Free standingparticles/cells in the fluid follow the flow pattern into the open well.(2) Once trapped, flow ceases in that localized region. (3) Solution isused to rinse off excess fluid and particles/cells. The device allowsfor the following analysis to be performed on the particle/cell, such asrepeated cycle of fluorescence imaging, bleaching, and analysis. Uponcompletion of analysis, (4) flow in the tubing is reversed to releasethe particle.

Example 4 Dual Capture eDAR

This example describes a “dual-capture” version of eDAR according to anaspect of the present disclosure. The dual-capture eDAR can separate twodifferent subpopulations of CTCs from the same human blood sample on thesame microfluidic device simultaneously. Those two subpopulations can betrapped separately on two different regions on the microfluidic chip,with a high recovery and purity, respectively.

FIG. 11 shows the general structure of the microfluidic device. Bloodsamples were pre-labeled with two kinds of antibodies conjugated withdifferent fluorescent tags. For example, the blood sample was labeledwith an epithelial marker, such as anti-EpCAM conjugated with PE, aswell as a mesenchymal marker, such as anti-EGFR or anti-vimentinconjugated with another fluorophore having a different wavelength ofemission compared to PE, such as FITC. The labeled blood sample wasinjected into the microfluidic chip, the CTCs with EpCAM expressioncould be detected using the line-confocal scheme with a peak in theyellow channel, and then an active sorting event was triggered tocollect that aliquot into the collection channel #1. Similarly, if thealiquot was ranked as positive to the mesenchymal markers, it was sortedto the collection channel #2. The two subpopulations were then trappedand enriched on the microfluidic chip separately. The filtration areawas built on the same design of microslits used in the second generationof eDAR.

The fluidic switching scheme is summarized as follows. When the aliquotswere ranked as negative to any marker applied, then both of thesolenoids in FIG. 11 were closed. If the pressure on the two sidechannels was balanced, the blood flowed to the bottom center channel,which was used to collect the waste. When the aliquots were ranked aspositive to epithelial marker only, solenoid #2 opened immediately sothe blood flow was pushed to the collection channel on the left (FIG.12B). After the aliquot was collected, solenoid #2 closed again, so theblood flow switched back to the center. When the aliquots were ranked aspositive to epithelial marker only, solenoid #1 opened immediately sothe blood flow was pushed to the right side (FIG. 12C). The responsetime for the two types of eDAR sorting events was about 2 to 3milliseconds.

FIG. 11 shows the general structure of the “dual-capture” eDAR. Labeledblood was introduced into the main channel on the top. Buffer wasflowing in the two side channels to control the hydrodynamic switchingof the blood flow using two solenoids. Two subpopulations of CTCs wereseparated and trapped on two different filtration areas on the samemicrofluidic chip.

FIG. 12 depicts bright field images of the three status of the bloodflow. A) The blood flowed into the waste collection channel, because thealiquots were ranked as negative to either markers. B) The blood wasswitched to the collection channel #1, and the first subpopulation ofCTCs transferred to there. C) The blood was switched to the collectionchannel #2, and the second subpopulation of CTCs transferred there.

Additional Aspects

Described herein are methods and apparatuses for analyzing particles,particularly particles in aliquots of a fluid sample wherein theparticles are analytes and the analytes are cells, in order to (1)identify a plurality of markers present on an analyte within a fluid,particularly by using a tag for at least each marker on the analyte anddetecting the signal emitted by each tag prior to removing the signalemitted by each tag and repeating the process of detecting and reducingthe signal, (2) isolate cells from a sample comprising a mixture offirst and second subtypes of cells, particularly by introducing thesample into a microfluidic chip comprising at least one channel fluidlyconnected to the set of tubing and to at least one chamber, (3)partition cells expressing a specific biomarker profile from a fluidsample, particularly using an apparatus that comprises a set of tubingconnected to a microfluidic chip that has at least one channel, (4)identify a plurality of markers present on an analyte, particularly bypartitioning a plurality of analytes using a substrate comprising aplurality of micro-cavities or micro-patches so as to contain no morethan one analyte in each micro-cavity or micro-patch, (5) detect aparticle in a fluid sample, particularly using a microfluidic chip withat least one sample input channel, at least one directional flowchannel, and at least two output channels, and an electro-actuated valvethat is located on a device that is not part of the microfluidic chip,(6) isolate an aliquot of a fluid sample within a microfluidic chip,particularly by assigning a value to the aliquot based on the presenceor absence of the rare particle and directing the flow of the aliquotbased on the assigned value by opening an electro-actuated valve locatedon a device that is external to the microfluidic chip; and (7) detect arare particle in a fluid sample using a device with at least two outputchannels, particularly with at least one of the two output channels isfluidly connected with an array of micro-apertures, and using the deviceto sort the one or more rare particles.

In some aspects, this disclosure provides methods for identifying aplurality of markers present on an analyte within a fluid, wherein themethod comprises: (a) detecting a signal from a first tag using a sourceof radiation, wherein the first tag is attached to a first structurethat binds to a first marker on the analyte; (b) partitioning theanalyte based on the presence of the first tag; reducing the level ofthe signal of the first tag; (c) contacting the analyte with a secondstructure that binds to a second marker, wherein the second structure isattached to a second tag; and (d) detecting the second tag. In someembodiments of these aspects, the signal of the first tag is reduced bygreater than 50%. In some aspects, the partitioning of step (a) is basedon the presence of the first tag and a third tag. In some aspects, thepartitioning of step (a) is performed with a microfluidic device. Insome aspects, the partitioning of step (a) and the detecting in step (b)occurs within the same microfluidic device. In some aspects, the analyteis a cell. In some aspects, the cell is a cancerous cell. In someaspects, the cancerous cell is a rare cell. In some aspects, the analyteis a circulating tumor cell. In some aspects, the cell is an immunecell, a fetal cell, a cell indicative of a disease remaining aftertreatment, or a stem cell. In some aspects, the fluid is selected fromthe group consisting of: whole blood, fractionated blood, serum, plasma,sweat, tears, ear flow, sputum, lymph, bone marrow suspension, lymph,urine, saliva, semen, vaginal flow, feces, transcervical lavage,cerebrospinal fluid, brain fluid, ascites, breast milk, vitreous humor,aqueous humor, sebum, endolymph, peritoneal fluid, pleural fluid,cerumen, epicardial fluid, and secretions of the respiratory, intestinaland genitourinary tracts. In some aspects, the fluid is whole blood. Insome aspects, the fluid is fractionated whole blood. In some aspects,the first tag is an antibody. In some aspects, the first tag is afluorophore. In some aspects, the first tag is a probe comprised of anucleic acid. In some aspects, the reduction of the signal in step (c)is accomplished by applying radiation to the analyte. In some aspects,the radiation is white light. In some aspects, the reduction of thesignal in step (c) is accomplished by applying a chemical to the firsttag. In some aspects, the chemical is a reducing agent. In some aspects,the reducing agent is dithiothreitol. In some aspects, the radiation isapplied using a laser. In some aspects, the radiation is applied using alight emitting diode (LED).

In some aspects, the method may further comprise imaging the signal fromthe first tag and the second tag. In some aspects, the analyte ispresent in a fluid, and the fluid is an aliquot of a larger volume offluid. In some aspects, the partitioning is performed semi-automaticallyor automatically. In some aspects, partitioning is performed byensemble-decision aliquot ranking. The In some aspects, each marker is abiomarker. In some aspects, the plurality of biomarkers is characterizedby an expression profile.

In some aspects, the method further comprises contacting the analytewith a buffer. In some aspects, the buffer contains a fixative. In someaspects, the buffer contains permeabilization agent. In some aspects,the buffer is a washing buffer. In some aspects, a flow cytometer is notused to partition the analyte. In some aspects, at the time of thedetecting in step a, the analyte is not a cell that is connected toadditional cells within a tissue.

Described herein are methods and compositions for isolating cells from acellular sample, in some aspects, the method of isolating cells from acellular sample may comprise a first cell subtype and a second cellsubtype that may further comprise, (a) introducing the sample into amicrofluidic chip via a set of tubing wherein the microfluidic chipcomprises, (i) at least one channel fluidly connected to the set oftubing; (ii) a detector configured to detect signals of cells within theat least one channel; and (iii) at least one chamber fluidly connectedto the at least one channel; (b) flowing a portion of the cellularsample past the detector; (c) using the detector to detect the presenceor absence of the first cell subtype within the portion of the cellularsample; (d) if the first cell subtype is detected within the portion ofthe cellular sample, directing an aliquot of the cellular sample intothe chamber, wherein the aliquot comprises the first cell subtype; and,(e) repeating steps (b)-(d), thereby isolating multiple aliquots in thechamber such that the chamber comprises greater than 80% of a totalnumber of first cell subtypes within the sample and less than 5% of atotal number of second cell subtypes within the sample.

In some aspects, provided herein are apparatuses for partitioning cellsexpressing a specific biomarker profile from a sample derived from afluid, wherein: the apparatus comprises a set of tubing connected to amicrofluidic chip that has at least one channel and a chamber; and theapparatus is capable of isolating the cells in the chamber, wherein,after isolation, the chamber comprises greater than 80% of the totalpopulation of cells in the sample expressing the specific biomarkerprofile and wherein, after isolation, the chamber comprises less than 5%of the total population of cells in the sample expressing a differentbiomarker profile. In some aspects, the isolation of the cellsexpressing a specific biomarker profile occurs in less than 20 minutes.In some aspects, the specific biomarker profile is present on less than5% of the cells in the sample of fluid. In some aspects, the fluid isblood. In some aspects, the fluid is fractionated whole blood. In someaspects, the fluid is the nucleated cell fraction of whole blood.

In some aspects, provided herein are methods for identifying a pluralityof markers present on an analyte, wherein the method comprises: (a)partitioning a plurality of analytes by flowing the analytes over asubstrate comprising a plurality of micro-cavities or micro-patches,wherein the majority of micro-cavities or micro-patches are capable ofcontaining not more than one analyte and wherein the micro-cavities ormicro-patches are located in a microfluidic device; (b) in themicro-cavities or micro-patches, contacting each analyte with a firststructure that is capable of binding to a first marker, wherein thefirst structure is connected to a first tag; (c) detecting a signal fromthe first tag; reducing the level of the signal of the first tag; (d)contacting the analyte with a second structure that binds to a secondmarker, wherein the second structure is connected to a second tag; and(e) detecting the second tag. In some aspects, the signal of the firsttag is reduced by greater than 50%. In some aspects, the contacting ofstep b is achieved by flowing a fluid comprising the first structurethrough a channel that is in fluid communication with the micro-cavitiesor micro-patch. In some aspects, following the contacting step of step(b), the method further comprises: contacting the analyte with a washbuffer. In some aspects, the analytes are cells. In some aspects, theanalytes are held in a fixed position within the micro-cavities by aforce generated by fluid flow, gravity, or adhesive forces. In someaspects, each analyte is connected to a micro-cavity or micro-patchthrough a molecular interaction. In some aspects, each analyte isconnected to a micro-cavity through a non-covalent bond. In someaspects, the non-covalent bond is a van der Waals interaction,electrostatic bond, hydrophobic bond or non-specific adsorption.

In some aspects, provided herein are devices for detecting a particle ina fluid sample, the device comprising: a microfluidic chip comprising atleast one sample input channel, at least one directional flow channel,and at least two output channels, wherein the at least one directionalflow channel intersects the sample input channel; an electro-actuatedvalve that is located on a device that is not part of the microfluidicchip, wherein the electro-actuated valve controls the flow of a liquidby controlling an input channel that intersects at least one directionalflow channel or at least one of the at least two output channels; atleast one detector capable of detecting one or more analytes in analiquot of the fluid sample; and a digital processor capable ofassigning a value to the aliquot based on the presence, absence,identity, composition, or quantity of analytes in the aliquot, whereinthe digital processor is in communication with the detector and theelectro-actuated valve. In some aspects, the electro-actuated valve is asolenoid valve. In some aspects, the electro-actuated valve controls theflow of the liquid in at least one directional flow channel. In someaspects, the electro-actuated valve is normally closed and wherein theelectro-activated valve opens after receiving a signal from thecomputer. In some aspects, the electro-actuated valve is normally openand wherein the electro-activated valve closes after receiving a signalfrom the computer. In some aspects, the at least one directional flowchannel comprises at least two ports and wherein the electro-actuatedvalve controls the flow of fluid through one of the ports. In someaspects, the device comprises a second detector. In some aspects, atleast one of the output channels is fluidly connected to a filter. Insome aspects, the electro-actuated valve directly controls the flow of aliquid in at least one directional flow channel. In some aspects, theelectro-actuated valve directly controls the flow of a liquid in achannel that feeds into at least one directional flow channel. In someaspects, the electro-actuated valve directly controls the flow of aliquid in only one directional flow channel. In some aspects, theelectro-actuated valve directly controls the flow of a liquid in one ofthe at least two output channels. In some aspects, the electro-actuatedvalve directly controls the flow of a liquid in a channel that feedsinto one of the at least two output channels. In some aspects, the atleast one directional flow channel intersects the at least two outputchannels at one or more junctions. In some aspects, the device comprisesa detector. In some aspects, the device comprises a confirmatory laser.In some aspects, the detector is located on at least one channel that isnot an output channel. In some aspects, the confirmatory laser islocated on at least one channel that is not an input channel. In someaspects, the electro-actuated valve is a piezo-electric valve.

In some aspects, provided herein are methods for isolating an aliquot ofa fluid sample within a microfluidic chip, wherein the aliquot comprisesa rare particle, the method comprising the steps of: detecting thepresence or absence of the rare particle in the aliquot; assigning avalue to the aliquot based on the presence or absence of the rareparticle; and directing the flow of the aliquot based on the assignedvalue by opening an electro-actuated valve, wherein the electro-actuatedvalve is located on a device that is external to the microfluidic chip.In some aspects, the microfluidic chip comprises a sample input channel,at least two output channels, and at least one directional flow channel,and wherein the electro-actuated valve controls the flow of fluid withinthe directional flow channel.

In some aspects, provided herein are devices for detecting a rareparticle in a fluid sample, the device comprising: at least a firstsample input channel; at least two output channels, wherein at least oneof the two output channels is fluidly connected with an array ofmicro-apertures; at least one detector capable of detecting one or morerare particles in an aliquot of the fluid sample; and a mechanism forsorting the one or more rare particles by directing the flow of aliquotscontaining the one or more rare particles through a first outputchannel. In some aspects, the mechanism directs the flow of the aliquotsinto a second output channel if the aliquot does not contain a rareparticle. In some aspects, the array of apertures is disposed betweenthe first sample input channel and the at least two output channels. Insome aspects, the array of apertures is in the same plane as the firstsample input channel and the output channels. In some aspects, the arrayof apertures is configured so that the rare particles cannot passthrough the apertures but at least one other particle is capable ofpassing through the apertures. In some aspects, the array of aperturescomprises greater than 1000 apertures. In some aspects, the mechanismfor sorting the rare particles comprises an electrode, a magneticelement, an acoustic element, or an electro-actuated element. In someaspects, the detector is selected from the group consisting of a camera,an electron multiplier, a charge-coupled device (CCD) image sensor, aphotomultiplier tube (PMT), an avalanche photodiode (APD), asingle-photon avalanche diode (SPAD), a silicon photomultiplier (SiPM),and a complementary metal oxide semiconductor (CMOS) image sensor.

In various aspects, methods are provided for identifying a plurality ofmarkers present on an analyte within a fluid, wherein the methodscomprise: (a) detecting a signal from a first tag using a source ofradiation, wherein the first tag is attached to a first structure thatbinds to a first marker on the analyte; (b) partitioning the analytebased on the presence of the first tag; (c) reducing the level of thesignal of the first tag; (d) contacting the analyte with a secondstructure that binds to a second marker, wherein the second structure isattached to a second tag; and (e) detecting the second tag.

In some aspects, the signal of the first tag is reduced by greater than50%. In other aspects, the partitioning of step (a) is based on thepresence of the first tag and a third tag. In further aspects, thepartitioning of step (a) is performed with a microfluidic device. Instill further aspects, the partitioning of step (a) and the detecting instep (b) occurs within the same microfluidic device. In some aspects,the analyte is a cell. In other aspects, the cell is a cancerous cell.In further aspects, the cancerous cell is a rare cell. In still furtheraspects, the analyte is a circulating tumor cell. In some aspects, thecell is an immune cell, a fetal cell, a cell indicative of a diseaseremaining after treatment, or a stem cell. In other aspects, the fluidis selected from the group consisting of: whole blood, fractionatedblood, serum, plasma, sweat, tears, ear flow, sputum, lymph, bone marrowsuspension, lymph, urine, saliva, semen, vaginal flow, feces,transcervical lavage, cerebrospinal fluid, brain fluid, ascites, breastmilk, vitreous humor, aqueous humor, sebum, endolympth, peritonealfluid, pleural fluid, cerumen, epicardial fluid, and secretions of therespiratory, intestinal and genitourinary tracts. In some aspects, thefluid is whole blood. In further aspects, the fluid is fractionatedwhole blood. In still further aspects, the first tag is an antibody. Insome aspects, the first tag is a fluorophore. In some aspects, the firsttag is a probe comprised of a nucleic acid. In other aspects, thereduction of the signal in step (c) is accomplished by applyingradiation to the analyte. In further aspects, the radiation is whitelight. In still further aspects, the reduction of the signal in step (c)is accomplished by applying a chemical to the first tag. In someaspects, the chemical is a reducing agent. In further aspects, thereducing agent is dithiothreitol. In some aspects, the radiation isapplied using a laser. In other aspects, the radiation is applied usinga light emitting diode (LED). In further aspects, the method furthercomprises imaging the signal from the first tag and the second tag. Instill further aspects, the analyte is present in a fluid, and the fluidis an aliquot of a larger volume of fluid. In some aspects, thepartitioning is performed semi-automatically or automatically. In otheraspects, the partitioning is performed by ensemble-decision aliquotranking. In some aspects, the each marker is a biomarker. In furtheraspects, the plurality of biomarkers is characterized by an expressionprofile. In still further aspects, the method further comprisescontacting the analyte with a buffer. In some aspects, the buffercontains a fixative. In other aspects, the buffer containspermeabilization agent. In further aspects, the buffer is a washingbuffer. In still further aspects, a flow cytometer is not used topartition the analyte. In some aspects, at the time of the detecting instep a, the analyte is not a cell that is connected to additional cellswithin a tissue.

In various aspects, methods are provided for isolating cells from acellular sample comprising a first cell subtype and a second cellsubtype comprising: (a) introducing the sample into a microfluidic chipvia a set of tubing wherein the microfluidic chip comprises (i) at leastone channel fluidly connected to the set of tubing; (ii) a detectorconfigured to detect signals of cells within the at least one channel;and (iii) at least one chamber fluidly connected to the at least onechannel; (b) flowing a portion of the cellular sample past the detector;(c) using the detector to detect the presence or absence of the firstcell subtype within the portion of the cellular sample; (d) if the firstcell subtype is detected within the portion of the cellular sample,directing an aliquot of the cellular sample into the chamber, whereinthe aliquot comprises the first cell subtype; and (e) repeating steps(b), (c), and (d), thereby isolating multiple aliquots in the chambersuch that the chamber comprises greater than 80% of a total number offirst cell subtypes within the sample and less than 5% of a total numberof second cell subtypes within the sample.

In various aspects, apparatuses are provided for partitioning cellsexpressing a specific biomarker profile from a sample derived from afluid, wherein: (a) the apparatuses comprise a set of tubing connectedto a microfluidic chip that has at least one channel and a chamber; and(b) the apparatuses are capable of isolating the cells in the chamber,wherein, after isolation, the chamber comprises greater than 80% of thetotal population of cells in the sample expressing the specificbiomarker profile and wherein, after isolation, the chamber comprisesless than 5% of the total population of cells in the sample expressing adifferent biomarker profile. In some aspects, the isolation of the cellsexpressing a specific biomarker profile occurs in less than 20 minutes.In other aspects, the specific biomarker profile is present on less than5% of the cells in the sample of fluid. In further aspects, the fluid isblood. In still further aspects, the fluid is fractionated whole blood.In some aspects, the fluid is the nucleated cell fraction of wholeblood.

In various aspects, methods are provided for identifying a plurality ofmarkers present on an analyte, wherein the methods comprise: (a)partitioning a plurality of analytes by flowing the analytes over asubstrate comprising a plurality of micro-cavities or micro-patches,wherein the majority of micro-cavities or micro-patches are capable ofcontaining not more than one analyte and wherein the micro-cavities ormicro-patches are located in a microfluidic device; (b) in themicro-cavities or micro-patches, contacting each analyte with a firststructure that is capable of binding to a first marker, wherein thefirst structure is connected to a first tag; (c) detecting a signal fromthe first tag; (d) reducing the level of the signal of the first tag;(e) contacting the analyte with a second structure that binds to asecond marker, wherein the second structure is connected to a secondtag; and (f) detecting the second tag.

In some aspects, the signal of the first tag is reduced by greater than50%. In other aspects, the contacting of step b is achieved by flowing afluid comprising the first structure through a channel that is in fluidcommunication with the micro-cavities or micro-patch. In furtheraspects, following the contacting step of step (b), the method furthercomprises: contacting the analyte with a wash buffer. In still furtheraspects, the analytes are cells. In some aspects, the analytes are heldin a fixed position within the micro-cavities by a force generated byfluid flow, gravity, or adhesive forces. In other aspects, each analyteis connected to a micro-cavity or micro-patch through a molecularinteraction. In further aspects, each analyte is connected to amicro-cavity through a non-covalent bond. In still further aspects, thenon-covalent bond is a van der Waals interaction, electrostatic bond,hydrophobic bond or non-specific adsorption.

In various aspects, devices are provided for detecting a particle in afluid sample, the devices comprising: (a) a microfluidic chip comprisingat least one sample input channel, at least one directional flowchannel, and at least two output channels, wherein the at least onedirectional flow channel intersects the sample input channel; (b) anelectro-actuated valve that is located on a device that is not part ofthe microfluidic chip, wherein the electro-actuated valve controls theflow of a liquid by controlling an input channel that intersects atleast one directional flow channel or at least one of the at least twooutput channels; (c) at least one detector capable of detecting one ormore analytes in an aliquot of the fluid sample; and (d) a digitalprocessor capable of assigning a value to the aliquot based on thepresence, absence, identity, composition, or quantity of analytes in thealiquot, wherein the digital processor is in communication with thedetector and the electro-actuated valve. In some aspects, theelectro-actuated valve is a solenoid valve. In other aspects, theelectro-actuated valve controls the flow of the liquid in at least onedirectional flow channel.

In further aspects, the electro-actuated valve is normally closed andwherein the electro-activated valve opens after receiving a signal fromthe computer. In still further aspects, the electro-actuated valve isnormally open and wherein the electro-activated valve closes afterreceiving a signal from the computer. In some aspects, the at least onedirectional flow channel comprises at least two ports and wherein theelectro-actuated valve controls the flow of fluid through one of theports. In other aspects, the device comprises a second detector. Infurther aspects, at least one of the output channels is fluidlyconnected to a filter.

In various aspects, methods are provided for isolating an aliquot of afluid sample within a microfluidic chip, wherein the aliquot comprises arare particle, the methods comprising the steps of: (a) detecting thepresence or absence of the rare particle in the aliquot; (b) assigning avalue to the aliquot based on the presence or absence of the rareparticle; and (c) directing the flow of the aliquot based on theassigned value by opening an electro-actuated valve, wherein theelectro-actuated valve is located on a device that is external to themicrofluidic chip.

In some aspects, the microfluidic chip comprises a sample input channel,at least two output channels, and at least one directional flow channel,and wherein the electro-actuated valve controls the flow of fluid withinthe directional flow channel.

In various aspects, devices are provided for detecting a rare particlein a fluid sample, the device comprising: (a) at least a first sampleinput channel; (b) at least two output channels, wherein at least one ofthe two output channels is fluidly connected with an array ofmicro-apertures; (c) at least one detector capable of detecting one ormore rare particles in an aliquot of the fluid sample; and (d) amechanism for sorting the one or more rare particles by directing theflow of aliquots containing the one or more rare particles through afirst output channel.

In some aspects, the mechanism directs the flow of the aliquots into asecond output channel if the aliquot does not contain a rare particle.In some aspects, the array of apertures is disposed between the firstsample input channel and the at least two output channels. In someaspects, the array of apertures is in the same plane as the first sampleinput channel and the output channels. In some aspects, the array ofapertures is configured so that the rare particles cannot pass throughthe apertures but at least one other particle is capable of passingthrough the apertures. In some aspects, the array of apertures comprisesgreater than 1000 apertures. In some aspects, the mechanism for sortingthe rare particles comprises an electrode, a magnetic element, anacoustic element, or an electro-actuated element. In some aspects, thedetector is selected from the group consisting of a camera, an electronmultiplier, a charge-coupled device (CCD) image sensor, aphotomultiplier tube (PMT), an avalanche photodiode (APD), asingle-photon avalanche diode (SPAD), a silicon photomultiplier (SiPM),and a complementary metal oxide semiconductor (CMOS) image sensor. Insome aspects, the electro-actuated valve directly controls the flow of aliquid in at least one directional flow channel. In some aspects, theelectro-actuated valve directly controls the flow of a liquid in achannel that feeds into at least one directional flow channel. In someaspects, the electro-actuated valve directly controls the flow of aliquid in only one directional flow channel. In some aspects, theelectro-actuated valve directly controls the flow of a liquid in one ofthe at least two output channels. In some aspects, the electro-actuatedvalve directly controls the flow of a liquid in a channel that feedsinto one of the at least two output channels. In some aspects, the atleast one directional flow channel intersects the at least two outputchannels at one or more junctions. In some aspects, the device comprisesa detector. In some aspects, the device comprises a confirmatory laser.In some aspects, the detector is located on at least one channel that isnot an output channel. In some aspects, the confirmatory laser islocated on at least one channel that is not an input channel. In someaspects, the electro-actuated valve is a piezo-electric valve.

1-110. (canceled)
 111. A method for identifying a plurality of markerspresent on an analyte, wherein the method comprises: (a) partitioning aplurality of analytes by flowing the plurality of analytes over asubstrate comprising a plurality of micro-cavities or micro-patches,wherein a majority of micro-cavities or micro-patches are configured tocontain not more than one analyte and wherein the plurality ofmicro-cavities or micro-patches are disposed in a microfluidic device;(b) in the plurality of micro-cavities or micro-patches, contacting ananalyte of the plurality of analytes with a first structure configuredto bind to a first marker, wherein the first structure is connected to afirst tag; (c) detecting a signal from the first tag; (d) reducing alevel of the signal of the first tag; (e) contacting the analyte with asecond structure that binds to a second marker, wherein the secondstructure is connected to a second tag; and (f) detecting the secondtag.
 112. The method of claim 111, wherein the contacting of step (b)comprises flowing a fluid comprising the first structure through achannel that is in fluid communication with the plurality ofmicro-cavities or micro-patches.
 113. The method of claim 111, wherein,following the contacting step of step (b), the method further comprises:contacting the analyte with a wash buffer.
 114. The method of claim 112,wherein the method is performed on a plurality of analytes isolated inthe plurality of micro-cavities or micro-patches.
 115. The method of anyone of claim 111, wherein reducing the level of the signal of the firsttag includes reducing the level of the signal of the first tag bygreater than 50%.
 116. The method of claim 111, wherein at least oneanalyte of the plurality of analytes is a cell.
 117. The method of claim111, wherein an analyte of the plurality of analytes is held in a fixedposition within the plurality of micro-cavities by a force generated byfluid flow, gravity, or adhesive forces.
 118. The method of claim 111,wherein an analyte of the plurality of analytes is connected to amicro-cavity or micro-patch of the plurality of micro-cavities ormicro-patches through a molecular interaction.
 119. The method of claim111, wherein an analyte of the plurality of analytes is connected to amicro-cavity through a non-covalent bond.
 120. The method of claim 119,wherein the non-covalent bond is chosen from a van der Waalsinteraction, an electrostatic bond, a hydrophobic bond, and anon-specific adsorption.
 121. A method for detecting a plurality ofmarkers present on an analyte, the method comprising: isolating ananalyte in a micro-cavity or in a micro-patch by flowing a fluid over asubstrate comprising the micro-cavity or micro-patch, wherein the fluidcomprises the analyte; contacting the analyte with a first tag, whereinthe analyte comprises a first marker, and wherein the first tag has anaffinity for the first marker; detecting a first signal emitted by thefirst tag, wherein the presence of the first signal indicates thepresence of the first marker; reducing an intensity of the first signal;contacting the analyte with a second tag, wherein the analyte comprisesa second marker, and wherein the second tag has an affinity for thesecond marker; and detecting a second signal emitted by the second tag,wherein the presence of the second signal indicates the presence of thesecond marker.
 122. The method of claim 121, wherein the contacting theanalyte with a first tag comprises flowing a fluid comprising the firsttag through a channel that is in fluid communication with themicro-cavity or micro-patch.
 123. The method of claim 121, wherein,following the contacting the analyte with a first tag, the methodfurther comprises: contacting the analyte with a wash buffer.
 124. Themethod of claim 121, wherein the method is performed on a plurality ofanalytes isolated in a plurality of micro-cavities or micro-patches.125. The method of claim 124, wherein at least one analyte of theplurality of analytes is a cell.
 126. The method of any one of claim121, wherein reducing the intensity of the signal of the first tagincludes reducing the intensity of the signal of the first tag bygreater than 50%.
 127. The method of claim 121, wherein the analyte isheld in a fixed position within the micro-cavity by a force generated byfluid flow, gravity, or adhesive forces.
 128. The method of claim 121,wherein the analyte is connected to a micro-cavity or micro-patchthrough a molecular interaction.
 129. The method of claim 121, whereinthe analyte is connected to a micro-cavity through a non-covalent bond.130. The method of claim 129, wherein the non-covalent bond is chosenfrom a van der Waals interaction, an electrostatic bond, a hydrophobicbond, or a non-specific adsorption.